Hydroxyl, keto, and glucuronide derivatives of 3-(4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile

ABSTRACT

The present invention provides hydroxyl, keto, and glucuronide derivatives of 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/250,387, filed Oct. 9, 2009, and U.S. Provisional Application No.61/316,218, filed Mar. 22, 2010. The disclosure of these documents areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention provides hydroxyl, keto, and glucuronidederivatives of3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile.

BACKGROUND OF THE INVENTION

Protein kinases (PKs) are a group of enzymes that regulate diverse,important biological processes including cell growth, survival anddifferentiation, organ formation and morphogenesis, neovascularization,tissue repair and regeneration, among others. Protein kinases exerttheir physiological functions through catalyzing the phosphorylation ofproteins (or substrates) and thereby modulating the cellular activitiesof the substrates in various biological contexts. In addition to thefunctions in normal tissues/organs, many protein kinases also play morespecialized roles in a host of human diseases including cancer. A subsetof protein kinases (also referred to as oncogenic protein kinases), whendysregulated, can cause tumor formation and growth, and furthercontribute to tumor maintenance and progression (Blume-Jensen P et al,Nature 2001, 411(6835):355-365). Thus far, oncogenic protein kinasesrepresent one of the largest and most attractive groups of proteintargets for cancer intervention and drug development.

The Janus Kinase (JAK) family plays a role in the cytokine-dependentregulation of proliferation and function of cells involved in immuneresponse. Currently, there are four known mammalian JAK family members:JAK1 (also known as Janus kinase-1), JAK2 (also known as Januskinase-2), JAK3 (also known as Janus kinase, leukocyte; JAKL; L-JAK andJanus kinase-3) and TYK2 (also known as protein-tyrosine kinase 2). TheJAK proteins range in size from 120 to 140 kDa and comprise sevenconserved JAK homology (JH) domains; one of these is a functionalcatalytic kinase domain, and another is a pseudokinase domainpotentially serving a regulatory function and/or serving as a dockingsite for STATs (Scott, Godshall et al. 2002, supra).

Blocking signal transduction at the level of the JAK kinases holdspromise for developing treatments for human cancers. Inhibition of theJAK kinases is also envisioned to have therapeutic benefits in patientssuffering from skin immune disorders such as psoriasis, and skinsensitization. Accordingly, inhibitors of Janus kinases or relatedkinases are widely sought and several publications report effectiveclasses of compounds. For example, certain JAK inhibitors, including(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrileshown below, are reported in U.S. Ser. No. 11/637,545 (US 2007/0135461),filed Dec. 12, 2006; U.S. Ser. No. 12/138,082 (US 2009/0181959), filedJul. 16, 2009; and certain metabolites of Compound I are reported inU.S. Ser. No. 12/137,883 (US 2008/0312258), filed Jun. 12, 2008, each ofwhich is incorporated herein by reference in its entirety.

Thus, new or improved agents which inhibit kinases such as Janus kinasesare continually needed for developing new and more effectivepharmaceuticals to treat cancer and other diseases. The metabolites,compositions and methods described herein are directed toward theseneeds and other ends.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, hydroxyl, keto, andglucuronide derivatives of a compound of Formula I:

or a pharmaceutically acceptable salt thereof.Thus, in one aspect, provided herein is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

n C—H groups are each independently replaced with C—OH; or,

one CH₂ group is independently replaced with a C═O; or,

one C—H group is replaced with:

two C—H groups are each independently replaced with C—OH and one C—Hgroup is replaced with:

n is 1, 2, 3, or 4;

provided that the compound is not selected from:

-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxocyclopentyl)propanenitrile;

and pharmaceutically acceptable salts thereof.

In one embodiment of Formula I,

the carbon atom alpha to the cyano group is not replaced with a C—OH orC═P group; and

the carbon atom beta to the cyano group is not replaced with a C—OHgroup.

In another embodiment of the compound, n C—H groups are eachindependently replaced with C—OH. In another embodiment, n is 1. In yetanother embodiment, n is 2. I still another embodiment, n is 3. Inanother embodiment, n is 4.

In another embodiment of Formula I, one CH₂ group is independentlyreplaced with a C═O. In still another embodiment, one C—H group isreplaced with

In another embodiment, one saturated C—H group is replaced with

In yet another embodiment, two C—H groups are each independentlyreplaced with C—OH and one C—H group is replaced with:

The present invention further provides compositions comprising compoundsdescribed herein, or a pharmaceutically acceptable salt thereof, and atleast one pharmaceutically acceptable carrier.

The present invention further provides methods of modulating an activityof JAK comprising contacting JAK with certain compounds describedherein, or pharmaceutically acceptable salt thereof. The presentinvention further provides methods of treating a disease in a patient,comprising administering to the patient a therapeutically effectiveamount of certain compounds described herein, or pharmaceuticallyacceptable salt thereof. In a particular embodiment, the disease isassociated with JAK activity. Such diseases include, for example,allograft rejection or graft versus host disease. The disease can alsobe an autoimmune disease, including, but not limited to, a skindisorder, multiple sclerosis, rheumatoid arthritis, juvenile arthritis,type I diabetes, lupus, inflammatory bowel disease, Crohn's disease,myasthenia gravis, immunoglobulin nephropathies, myocarditis, orautoimmune thyroid disorder. The autoimmune disease can also be bullousskin disorder, e.g., pemphigus vulgaris (PV) or bullous pemphigoid (BP).The skin disorder can be atopic dermatitis, psoriasis, skinsensitization, skin irritation, skin rash, contact dermatitis orallergic contact sensitization.

In another embodiment, the disease is a viral disease. Examples of viraldiseases that can be treated by the compounds described herein includeEpstein Barr Virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLV 1,Varicell-Zoster Virus (VZV) or Human Papilloma Virus (HPV).

In another embodiment, the disease is cancer, e.g., a solid tumor. Thecancer to be treated can be prostate cancer, renal cancer, hepaticcancer, breast cancer, lung cancer, thyroid cancer, Kaposi's sarcoma,Castleman's disease or pancreatic cancer. In a particular embodiment,the cancer is prostate cancer. The cancer can be hematological. Thecancer can also be a lymphoma, leukemia, or multiple myeloma. In anotherembodiment, the cancer is a skin cancer, e.g., cutaneous T-cell lymphomaor cutaneous B-cell lymphoma. In another embodiment, the cancer ismultiple myeloma. The disease to be treated can also be fatigueresulting from or associated with cancer, or anorexia or cachexiaresulting from or associated with cancer.

In another embodiment, the disease to be treated is a myeloproliferativedisorder, e.g., polycythemia vera (PV), essential thrombocythemia (ET),myeloid metaplasia with myelofibrosis (MMM), chronic myelogenousleukemia (CML), chronic myelomonocytic leukemia (CMML),hypereosinophilic syndrome (HES), or systemic mast cell disease (SMCD).

In another embodiment, the disease to be treated is an inflammatorydisease. The inflammatory disease can be an inflammatory disease of theeye, e.g., iritis, uveitis, scleritis, or conjunctivitis. Theinflammatory disease can be an inflammatory disease of the respiratorytract, e.g., the upper respiratory tract or the lower respiratory tract.The inflammatory disease can be an inflammatory myopathy or myocarditis.

In another embodiment, the disease is ischemia reperfusion or related toan ischemic event.

DETAILED DESCRIPTION

The present invention provides, inter alia, hydroxyl, keto, andglucuronide derivatives of3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile.In some embodiments, the compound is a metabolite of compound I. In someembodiments, the compound is an active metabolite which may modulate theactivity of one or more JAKs and may be useful, for example, in thetreatment of diseases associated with JAK expression or activity. Insome embodiments, the level of a metabolite compound described herein ismeasured and profiled in order to aid a practitioner in the adjustmentof dosage levels of the compound of Formula I.

Accordingly, the present invention provides a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

n C—H groups are each independently replaced with C—OH; or,

one CH₂ group is independently replaced with a C═O; or,

one C—H group is replaced with:

two C—H groups are each independently replaced with C—OH and one C—Hgroup is replaced with:

n is 1, 2, 3, or 4;

provided that the compound is not selected from:

-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxocyclopentyl)propanenitrile;

and pharmaceutically acceptable salts thereof.

In some embodiments, n C—H groups are each independently replaced withC—OH. In some embodiments, n is 1. In some embodiments, n is 2. In someembodiments, n is 3. In some embodiments, n is 4.

In some embodiments, one CH₂ group is independently replaced with a C═O.

In some embodiments, one C—H group is replaced with

In some embodiments, one saturated C—H group is replaced with

In some embodiments, two C—H groups are each independently replaced withC—OH and one C—H group is replaced with:

In some embodiments:

-   -   the carbon atom alpha to the cyano group is not replaced with a        C—OH, C═O, or

group; and

-   -   the carbon atom beta to the cyano group is not replaced with a        C—OH or

group.In some embodiments:

-   -   the carbon atom alpha to the cyano group is not replaced with a        C—OH or C═O group; and    -   the carbon atom beta to the cyano group is not replaced with a        C—OH group.

In another embodiment, the present invention provides a compound ofFormula II:

or a pharmaceutically acceptable salt thereof, wherein:

1, 2, 3, or 4 of carbons a, b, c, d, e, f, g, h, i, j, k or m are eachindependently substituted with OH; or,

one of carbons h, i, j, k, or m are independently substituted with ═O;or,

one of carbons a, b, c, d, e, f, g, h, i, j, k or m are substitutedwith:

two of carbons a, b, c, d, e, f, g, h, i, j, k or in are eachindependently substituted with OH and:

provided that the compound is not selected from:

-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxocyclopentyl)propanenitrile;

and pharmaceutically acceptable salts thereof.

In some embodiments of Formula II, 1, 2, 3, or 4 of carbons a, b, c, d,e, f, g, h, j, k or m are each independently substituted with OH. Insome embodiments, one of carbons a, b, c, d, e, f, g, h, i, j, k or inare substituted with OH. In some embodiments, 2 of carbons a, b, c, d,e, f, g, h, i, j, k or m are each independently substituted with OH. Insome embodiments, 3 of carbons a, b, c, d, e, f, g, h, i, j, k or m areeach independently substituted with OH. In some embodiments, 4 ofcarbons a, b, c, d, e, f, g, h, i, j, k or in are each independentlysubstituted with OH.

In some embodiments, one of carbons h, i, j, k, or m are substitutedwith ═O.

In some embodiments, one of carbons a, b, c, d, e, f, g, h, i, j, k or mare substituted with:

In some embodiments, one of carbons f, g, h, i, j, k or m aresubstituted with:

In some embodiments, two of carbons a, b, c, d, e, f, g, h, i, j, k or mare each independently substituted by OH and one of carbons a, b, c, d,e, f, g, h, i, j, k or m are substituted with:

In some embodiments:

carbon m is not substituted with a OH, ═O, or

group; and

carbon f is not substituted with a OH or

group.

In some embodiments:

carbon m is not substituted with a OH or ═O group; and

carbon f is not substituted with a OH group.

Each of the aforementioned embodiments assumes that the rules for propervalency are adhered to.

In some embodiments, the compound is selected from:

-   6-(3-(1-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-2-cyanoethyl)cyclopentyloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic    acid;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1,2-dihydroxycyclopentyl)propanenitrile;    and-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;

or a pharmaceutically acceptable salt of any of the aforementioned.

In some embodiments, the compound is selected from:

-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-3-hydroxypropanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-2-hydroxypropanenitrile;-   3-cyclopentyl-3-(5-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;-   3-cyclopentyl-3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;-   3-cyclopentyl-3-(4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;-   3-cyclopentyl-3-(4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;-   3-cyclopentyl-3-(4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1,2-dihydroxycyclopentyl)propanenitrile;-   3-cyclopentyl-3-(4-(5,6-dihydroxy-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;    and-   6-(4-(1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic    acid;

or a pharmaceutically acceptable salt of any of the aforementioned.

In some embodiments, the compounds described herein can include thecompounds shown in the charts 1-7 below, and enantiomers, diastereomers,and racemates thereof.

Certain metabolites are indicated in Table 1 below. Structures areintended to encompass all possible stereoisomers.

TABLE 1 Reference* Name Structure Metabolite 1 (3/8)6-(3-(1-(4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-2-cyanomethyl)cyclopentyloxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid

Metabolite 2 (31) 3-cyclopentyl-3-(4-(2,6-dioxo-3,5,6,7-tetrahydro-2H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile

Metabolite 3 (32) 3-cyclopentyl-3-(4-(2-hydroxy-5-oxo-6,7-dihydro-5H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile

Metabolite 4 (35) 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1,2- dihydroxycyclopentyl)propanenitrile

Metabolite 5 (36) 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1- hydroxycyclopentyl)propanenitrile

Metabolite 6 (37) 3-cyclopentyl-3-(4-(6-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin- 4-yl)-1H-pyrazol-1-yl)propanenitrile

Metabolite 7 (38) 3-(3-hydroxycyclopentyl)-3-(4-(6-oxo-6,7-dihydro-5H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile

Metabolite 8 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3- hydroxycyclopentyl)propanenitrile

Metabolite 9 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2- hydroxycyclopentyl)propanenitrile

*The numbers in parentheses refer to the compound numbers in Table 2(infra)

Compounds described herein are metabolites of(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile.The metabolites of the invention were isolated from human, rat or dogurine samples collected from pharmacokinetic and toxicokinetic studiesof the JAK inhibitor(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile(Compound I). Certain metabolites may be JAK inhibitors, and can haveadvantageous properties related to significantly higher free fractionsand higher metabolic stability in human microsomes compared withCompound I. The present metabolites may desirably have a longerelimination half-life in humans than does Compound I.

In some embodiments, the metabolites of the invention are substantiallyisolated. By “substantially isolated” is meant that the compound is atleast partially or substantially separated from the environment in whichit was formed or detected. Partial separation can include, for example,a composition enriched in the compound of the invention. Substantialseparation can include compositions containing at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 97%, or at least about 99% byweight of the metabolite.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present invention can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal ofPharmaceutical Science, 66, 2 (1977), each of which is incorporatedherein by reference in its entirety.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The compounds described herein are asymmetric (e.g., having one or morestereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Methods on howto prepare optically active forms from optically active startingmaterials are known in the art, such as by resolution of racemicmixtures or by stereoselective synthesis.

Compounds described herein also include all isotopes of atoms occurringin the metabolites. Isotopes include those atoms having the same atomicnumber but different mass numbers. For example, isotopes of hydrogeninclude tritium and deuterium. The compounds may also include solvatesand hydrates of the compounds or salt forms.

The term, “compound,” as used herein is meant to include allstereoisomers, geometric iosomers, tautomers, and isotopes of thestructures depicted.

Synthesis

Compounds described herein, including salts thereof, can be preparedusing known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes.

The reactions for preparing compounds described herein can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds described herein can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley & Sons,Inc., New York (1999), which is incorporated herein by reference in itsentirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), ormass spectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Compounds described herein can be prepared according to numerouspreparatory routes known in the literature. For example, compoundsdescribed herein may be made by processes analogous to those describedin U.S. Ser. No. 11/637,545 (US 2007/0135461), filed Dec. 12, 2006; U.S.Ser. No. 12/138,082 (US 2009/0181959), filed Jul. 16, 2009; and U.S.Ser. No. 12/137,883 (US 2008/0312258), filed Jun. 12, 2008, each ofwhich is incorporated herein by reference in its entirety. Examplesynthetic methods for preparing compounds described herein are providedin the Schemes below.

As shown in Scheme 1, synthesis of compound 32 starts with 4-chloropyrrolopyrimidine S1. The Suzuki coupling of S1 with the pyrazoleboronate S2 under basic conditions in the presence of Pd(0) catalyst mayprovide the tricyclic compound S3 which is subsequently convertedselectively to the iodo compound S4 using N-iodosuccinimide. The iodocompound S4 is transformed to the corresponding acetate S5. Treatment ofS5 with 4M hydrogen bromide in acetic acid provides S6 with the desiredoxidation in the pyrrole ring along with the deprotection of the methoxygroup to the free hydroxyl. The cyano group which was hydrolyzed to theamide under these conditions is reinstalled in 32 by treatment of S6with trichloroacetyl chloride and triethylamine to effect thedehydration.

The mono-hydroxylated compound 42 (metabolite 8) has been reported inU.S. Ser. No. 12/137,883, filed Jun. 12, 2008. Further, compounds havinga hydroxyl group on the cyclopentyl ring may also be synthesized as inScheme 2. For example, the commercially available racemic S14 in whichthe alcohol is protected followed by reduction of the carboxylic esterto the aldehyde followed by an olefination of the aldehyde to provideS17. Conjugate addition of S9 to S17 under basic conditions provides S18from which the protecting groups on the alcohol and pyrrole ring areremoved sequentially to provide a mixture of diastereomers which arethen separated using multiple chiral HPLC columns to provide the fourdiastereomers of S19. In some embodiments, the present inventionprovides a process of making compound 42, or a pharmaceuticallyacceptable salt thereof, comprising one or more of the steps in Scheme2.

Another example of the mono-hydroxylated compound 36 is obtained by thesequence shown in Scheme 3. The protected cycanohydrin S31 can bereduced with DIBAL to the corresponding aldehyde followed by theolefination with the phosphonate such as S7 to provide the crotontirileS33. Conjugate addition of S9 to S33 under basic conditions provides S34from which the protecting group on the pyrrole ring is removed toprovide a mixture of diastereomers which can then be separated usingmultiple chiral HPLC columns to provide the individual stereoisomers of36.

The dihydroxylated metabolites may be obtained in a similar fashion asshown in Scheme 4: cyclopentene carboxaldehyde S6A may be treateddirectly with the ylid S7 to give the crotonitrile derivative S8. Thenitrile S8 then can be reacted with the pyrazole S9 in the presence of abase such as DBU to give S10 as a mixture of diastereomers, which can bedihydroxylated with osmium tetroxide to provide the cis-alcoholscis-S12A, after removal of the SEM group. The individual stereoisomersof this mixture (cis-S12A) may be separated by chiral chromatography togive the enantiomerically pure alcohols. The trans-S12A may be obtainedby first epoxidizing the olefin with m-CPBA followed by opening up theepoxide under acidic conditions. The individual stereoisomers of thismixture (trans-S12A) may be separated by chiral chromatography to givethe enantiomerically pure alcohols. The same synthetic route may beadapted to obtain the isomeric S12B and S12C by replacing the startingaldehyde S6A with S6B and S6C.

A combination of the methods described above along with those describedin U.S. patent application Ser. No. 11/637,545, filed Dec. 12, 2006; andin U.S. patent application Ser. No. 12/137,883, filed Jun. 12, 2008,which are incorporated herein in its entirety, may be utilized to obtainthe trihydroxy compounds of the invention. For example, the hydroxy anddihydroxy compounds described herein may be oxidized under Swernoxidation conditions described in U.S. patent application Ser. No.12/137,883, filed Jun. 12, 2008. The glucuronides of metabolitescontaining hydroxyl groups may be synthesized according to the procedureof Suzuki et al. (Bioorg. Med. Chem. Lett. (1999), 9(5), 659-662 whichis incorporated herein in its entirety).

Methods

Certain compounds described herein may modulate activity of one or moreJanus kinases (JAKs). The term “modulate” is meant to refer to anability to increase or decrease the activity of one or more members ofthe JAK family of kinases. Accordingly, certain compounds describedherein can be used in methods of modulating a JAK by contacting the JAKwith any one or more of the compounds or compositions described herein.In some embodiments, certain compounds may act as inhibitors of one ormore JAKs. In some embodiments, certain compounds described herein canact to stimulate the activity of one or more JAKs. In furtherembodiments, certain compounds may be used to modulate activity of a JAKin an individual in need of modulation of the receptor by administeringa modulating amount of a compound of the invention.

JAKs to which compounds bind and/or modulate may include any member ofthe JAK family. In some embodiments, the JAK is JAK1, JAK2, JAK3 orTYK2. In some embodiments, the JAK is JAK1 or JAK2. In some embodiments,the JAK is JAK2. In some embodiments, the JAK is JAK3.

In some embodiments, active compounds may be selective. By “selective”is meant that the compound binds to or inhibits a JAK with greateraffinity or potency, respectively, compared to at least one other JAK(e.g., selective inhibitors of JAK1 or JAK2 over JAK3 and/or TYK2). Insome embodiments, selective means selective inhibition of JAK2 (e.g.,over JAK1, JAK3 and TYK2). Without wishing to be bound by theory,because inhibitors of JAK3 can lead to immunosuppressive effects, acompound which is selective for JAK2 over JAK3 and which is useful inthe treatment of cancer (such as multiple myeloma, for example) canoffer the additional advantage of having fewer immunosuppressive sideeffects. Selectivity may be at least about 5-fold, at least about10-fold, at least about 20-fold, at least about 50-fold, at least about100-fold, at least about 200-fold, at least about 500-fold or at leastabout 1000-fold. Selectivity may be measured by methods routine in theart. In some embodiments, selectivity may be tested at the Km of eachenzyme. In some embodiments, selectivity for JAK2 over JAK3 can bedetermined by the cellular ATP concentration.

Another aspect of the present invention pertains to methods of treatinga JAK-associated disease or disorder in an individual (e.g., patient) byadministering to the individual in need of such treatment atherapeutically effective amount or dose of a certain compound describedherein or a pharmaceutical composition thereof. A JAK-associated diseasecan include any disease, disorder or condition that is directly orindirectly linked to expression or activity of the JAK, includingoverexpression and/or abnormal activity levels. A JAK-associated diseasecan also include any disease, disorder or condition that can beprevented, ameliorated, or cured by modulating JAK activity.

Examples of JAK-associated diseases include diseases involving theimmune system including, for example, organ transplant rejection (e.g.,allograft rejection and graft versus host disease).

Further examples of JAK-associated diseases include autoimmune diseasessuch as multiple sclerosis, rheumatoid arthritis, juvenile arthritis,type I diabetes, lupus, psoriasis, inflammatory bowel disease,ulcerative colitis, Crohn's disease, myasthenia gravis, immunoglobulinnephropathies, autoimmune thyroid disorders, and the like. In someembodiments, the autoimmune disease is an autoimmune bullous skindisorder such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).

Further examples of JAK-associated diseases include allergic conditionssuch as asthma, food allergies, atopic dermatitis and rhinitis. Furtherexamples of JAK-associated diseases include viral diseases such asEpstein Barr Virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLV 1,Varicella-Zoster Virus (VZV) and Human Papilloma Virus (HPV).

Further examples of JAK-associated diseases or conditions include skindisorders such as psoriasis (for example, psoriasis vulgaris), atopicdermatitis, skin rash, skin irritation, skin sensitization (e.g.,contact dermatitis, allergic contact dermatitis, or allergic contactsensitization). For example, certain substances including somepharmaceuticals when topically applied can cause skin sensitization. Insome embodiments, co-administration or sequential administration of atleast one JAK inhibitor together with the agent causing unwantedsensitization can be helpful in treating such unwanted sensitization ordermatitis. In some embodiments, the skin disorder is treated by topicaladministration of at least one JAK inhibitor.

In further embodiments, the JAK-associated disease is cancer includingthose characterized by solid tumors (e.g., prostate cancer, renalcancer, hepatic cancer, pancreatic cancer, gastric cancer, breastcancer, lung cancer, cancers of the head and neck, thyroid cancer,glioblastoma, Kaposi's sarcoma, Castleman's disease, melanoma etc.),hematological cancers (e.g., lymphoma, leukemia such as acutelymphoblastic leukemia, acute myelogenous leukemia (AML) or multiplemyeloma), and skin cancer such as cutaneous T-cell lymphoma (CTCL) andcutaneous B-cell lymphoma. Example cutaneous T-cell lymphomas includeSezary syndrome and mycosis fungoides.

JAK-associated diseases can further include those characterized byexpression of: JAK2 mutants such as those having at least one mutationin the pseudo-kinase domain (e.g., JAK2V617F); JAK2 mutants having atleast one mutation outside of the pseudo-kinase domain; JAK1 mutants;JAK3 mutants; erythropoietin receptor (EPOR) mutants; or deregulatedexpression of CRLF2.

JAK-associated diseases can further include myeloproliferative disorders(MPDs) such as polycythemia vera (PV), essential thrombocythemia (ET),myeloid metaplasia with myelofibrosis (MMM), chronic myelogenousleukemia (CML), chronic myelomonocytic leukemia (CMML),hypereosinophilic syndrome (HES), systemic mast cell disease (SMCD), andthe like.

Further JAK-associated diseases include inflammation and inflammatorydiseases. Example inflammatory diseases include sarcoidosis,inflammatory diseases of the eye (e.g., dry eye, iritis, uveitis,scleritis, conjunctivitis, or related disease), inflammatory diseases ofthe respiratory tract (e.g., the upper respiratory tract including thenose and sinuses such as rhinitis or sinusitis or the lower respiratorytract including bronchitis, chronic obstructive pulmonary disease, andthe like), inflammatory myopathy such as myocarditis, and otherinflammatory diseases. Other inflammatory diseases treatable by JAKinhibitors include systemic inflammatory response syndrome (SIRS) andseptic shock.

As used herein, “dry eye disorder” is intended to encompass the diseasestates summarized in a recent official report of the Dry Eye Workshop(DEWS), which defined dry eye as “a multifactorial disease of the tearsand ocular surface that results in symptoms of discomfort, visualdisturbance, and tear film instability with potential damage to theocular surface. It is accompanied by increased osmolarity of the tearfilm and inflammation of the ocular surface.” Lemp, “The Definition andClassification of Dry Eye Disease: Report of the Definition andClassification Subcommittee of the International Dry Eye Workshop”, TheOcular Surface, 5(2), 75-92 April 2007, which is incorporated herein byreference in its entirety. In some embodiments, the dry eye disorder isselected from aqueous tear-deficient dry eye (ADDE) or evaporative dryeye disorder, or appropriate combinations thereof. In some embodiments,the dry eye disorder is Sjogren syndrome dry eye (SSDE). In someembodiments, the dry eye disorder is non-Sjogren syndrome dry eye(NSSDE).

In a further aspect, the present invention provides a method of treatingconjunctivitis, uveitis (including chronic uveitis), chorioditis,retinitis, cyclitis, sclieritis, episcleritis, or iritis; treatinginflammation or pain related to corneal transplant, LASIK (laserassisted in situ keratomileusis), photorefractive keratectomy, or LASEK(laser assisted sub-epithelial keratomileusis); inhibiting loss ofvisual acuity related to corneal transplant, LASIK, photorefractivekeratectomy, or LASEK; or inhibiting transplant rejection in a patientin need thereof, comprising administering to the patient atherapeutically effective amount of the compound of the invention, or apharmaceutically acceptable salt thereof.

JAK inhibitors can further be used to treat ischemia reperfusioninjuries or a disease or condition related to an inflammatory ischemicevent such as stroke or cardiac arrest. JAK inhibitors can further beused to treat anorexia, cachexia, or fatigue such as that resulting fromor associated with cancer. JAK inhibitors can further be used to treatrestenosis, sclerodermitis, or fibrosis. JAK inhibitors can further beused to treat conditions associated with hypoxia or astrogliosis suchas, for example, diabetic retinopathy, cancer, or neurodegeneration.See, e.g., Dudley, A. C. et al. Biochem. J. 2005, 390(Pt 2):427-36 andSriram, K. et al. J. Biol. Chem. 2004, 279(19):19936-47. Epub 2004 Mar.2.

JAK inhibitors can further be used to treat gout and increased prostatesize due to, e.g., benign prostatic hypertrophy or benign prostatichyperplasia.

Further JAK-associated diseases include bone resorption diseases such asosteoporosis, osteoarthritis. Bone resorption can also be associatedwith other conditions such as hormonal imbalance and/or hormonaltherapy, autoimmune disease (e.g. osseous sarcoidosis), or cancer (e.g.myeloma). The reduction of the bone resorption due to the JAK inhibitorscan be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, or about 90%.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a JAK with a compound includes the administrationof a compound of the present invention to an individual or patient, suchas a human, having a JAK, as well as, for example, introducing acompound into a sample containing a cellular or purified preparationcontaining the JAK.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) preventing the disease; for example, preventing a disease,condition or disorder in an individual who may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease; (2) inhibiting thedisease; for example, inhibiting a disease, condition or disorder in anindividual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder; and (3)ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology) such as decreasing theseverity of disease.

The levels of metabolites in a patient after administration of compoundI in an individual can be measured and profiled. Such metabolite profilecan then be used to adjust the dosage regimens (e.g., for administrationof compound I) in that individual. For example, faster clearance ofcompound I as shown by levels of various metabolites after a given timeperiod may mean that initial dosages should be adjusted upwards.

Combination Therapies

One or more additional pharmaceutical agents such as, for example,chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, as well as Bcr-Abl, Flt-3, RAF and FAK kinaseinhibitors such as, for example, those described in WO 2006/056399, orother agents can be used in combination with certain compounds describedherein for treatment of JAK-associated diseases, disorders orconditions. The one or more additional pharmaceutical agents can beadministered to a patient simultaneously or sequentially.

Example chemotherapeutics include proteosome inhibitors (e.g.,bortezomib), thalidomide, revlimid, and DNA-damaging agents such asmelphalan, doxorubicin, cyclophosphamide, vincristine, etoposide,carmustine, and the like.

Example steroids include coriticosteroids such as dexamethasone orprednisone.

Example Bcr-Abl inhibitors include the compounds, and pharmaceuticallyacceptable salts thereof, of the genera and species disclosed in U.S.Pat. No. 5,521,184, WO 04/005281, and U.S. Ser. No. 60/578,491.

Example suitable Flt-3 inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 03/037347, WO03/099771, and WO 04/046120.

Example suitable RAF inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO05/028444.

Example suitable FAK inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 04/080980, WO04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402.

In some embodiments, one or more compounds described herein may be usedin combination with one or more other kinase inhibitors includingimatinib, particularly for treating patients resistant to imatinib orother kinase inhibitors.

In some embodiments, one or more compounds can be used in combinationwith a chemotherapeutic in the treatment of cancer, such as multiplemyeloma, and may improve the treatment response as compared to theresponse to the chemotherapeutic agent alone, without exacerbation ofits toxic effects. Examples of additional pharmaceutical agents used inthe treatment of multiple myeloma, for example, can include, withoutlimitation, melphalan, melphalan plus prednisone [MP], doxorubicin,dexamethasone, and Velcade (bortezomib). Further additional agents usedin the treatment of multiple myeloma include Bcr-Abl, Flt-3, RAF and FAKkinase inhibitors. Additive or synergistic effects are desirableoutcomes of combining a compound of the present invention with anadditional agent. Furthermore, resistance of multiple myeloma cells toagents such as dexamethasone may be reversible upon treatment with acompound of the present invention. The agents can be combined with theJAK inhibitor in a single or continuous dosage form, or the agents canbe administered simultaneously or sequentially as separate dosage forms.

In some embodiments, a corticosteroid such as dexamethasone isadministered to a patient in combination with at least one JAK inhibitorwhere the dexamethasone is administered intermittently as opposed tocontinuously.

In some further embodiments, combinations of one or more compounds withother therapeutic agents can be administered to a patient prior to,during, and/or after a bone marrow transplant or stein cell transplant.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds described herein can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. In some embodiments,the composition is suitable for oral administration. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal, intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds described hereinin combination with one or more pharmaceutically acceptable carriers(excipients). In making the compositions of the invention, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

The active ingredient may be milled using known milling procedures suchas wet milling to obtain a particle size appropriate for tabletformation and for other formulation types. Finely divided(nanoparticulate) preparations of the active ingredient can be preparedby processes known in the art, for example see International PatentApplication No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

In some embodiments, the compositions of the invention contain fromabout 5 mg to about 50 mg of the active ingredient. One having ordinaryskill in the art will appreciate that this embodies compounds orcompositions containing about 5 mg to about 10 mg, about 10 mg to about15 mg, about 15 mg to about 20 mg, about 20 mg to about 25 mg, about 25mg to about 30 mg, about 30 mg to about 35 mg, about 35 mg to about 40mg, about 40 mg to about 45 mg, or about 45 mg to about 50 mg of theactive ingredient.

In some embodiments, the compositions of the invention contain fromabout 50 mg to about 500 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodies compoundsor compositions containing about 50 mg to about 100 mg, about 100 mg toabout 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250mg, about 250 mg to about 300 mg, about 350 mg to about 400 mg, or about450 mg to about 500 mg of the active ingredient.

In some embodiments, the compositions of the invention contain fromabout 500 mg to about 1,000 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodies compoundsor compositions containing about 500 mg to about 550 mg, about 550 mg toabout 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700mg, about 700 mg to about 750 mg, about 750 mg to about 800 mg, about800 mg to about 850 mg, about 850 mg to about 900 mg, about 900 mg toabout 950 mg, or about 950 mg to about 1,000 mg of the activeingredient.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions can beincorporated for administration orally or by injection include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face masks tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds can vary according to, forexample, the particular use for which the treatment is made, the mannerof administration of the compound, the health and condition of thepatient, and the judgment of the prescribing physician. The proportionor concentration of a compound in a pharmaceutical composition can varydepending upon a number of factors including dosage, chemicalcharacteristics (e.g., hydrophobicity), and the route of administration.For example, the compounds can be provided in an aqueous physiologicalbuffer solution containing about 0.1 to about 10% w/v of the compoundfor parenteral administration. Some typical dose ranges are from about 1μg/kg to about 1 g/kg of body weight per day. In some embodiments, thedose range is from about 0.01 mg/kg to about 100 mg/kg of body weightper day. The dosage is likely to depend on such variables as the typeand extent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, formulation of the excipient, and its route ofadministration. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which arelisted hereinabove.

In some embodiments, the compound, or pharmaceutically acceptable saltthereof, is administered as an ophthalmic composition. Accordingly, insome embodiments, the methods comprise administration of the compound,or pharmaceutically acceptable salt thereof, and an ophthalmicallyacceptable carrier. In some embodiments, the ophthalmic composition is aliquid composition, semi-solid composition, insert, film, microparticlesor nanoparticles.

In some embodiments, the ophthalmic composition is a liquid composition.In some embodiments, the ophthalmic composition is a semi-solidcomposition. In some embodiments, the ophthalmic composition is antopical composition. The topical compositions include, but are notlimited to liquid and semi-solid compositions. In some embodiments, theophthalmic composition is a topical composition. In some embodiments,the topical composition comprises aqueous solution, an aqueoussuspension, an ointment or a gel. In some embodiments, the ophthalmiccomposition is topically applied to the front of the eye, under theupper eyelid, on the lower eyelid and in the cul-de-sac. In someembodiments, the ophthalmic composition is sterilized. The sterilizationcan be accomplished by known techniques like sterilizing filtration ofthe solution or by heating of the solution in the ampoule ready for use.The ophthalmic compositions of the invention can further containpharmaceutical excipients suitable for the preparation of ophthalmicformulations. Examples of such excipients are preserving agents,buffering agents, chelating agents, antioxidant agents and salts forregulating the osmotic pressure.

As used herein, the term “ophthalmically acceptable carrier” refers toany material that can contain and release the compound, orpharmaceutically acceptable salt thereof, and that is compatible withthe eye. In some embodiments, the ophthalmically acceptable carrier iswater or an aqueous solution or suspension, but also includes oils suchas those used to make ointments and polymer matrices such as used inocular inserts. In some embodiments, the composition may be an aqueoussuspension comprising the compound, or pharmaceutically acceptable saltthereof. Liquid ophthalmic compositions, including both ointments andsuspensions, may have a viscosity that is suited for the selected routeof administration. In some embodiments, the ophthalmic composition has aviscosity in the range of from about 1,000 to about 30,000 centipoise.

In some embodiments, the ophthalmic compositions may further compriseone or more of surfactants, adjuvants, buffers, antioxidants, tonicityadjusters, preservatives (e.g., EDTA, BAK (benzalkonium chloride),sodium chlorite, sodium perborate, polyquaterium-1), thickeners orviscosity modifiers (e.g., carboxymethyl cellulose, hydroxymethylcellulose, polyvinyl alcohol, polyethylene glycol, glycol 400, propyleneglycol hydroxymethyl cellulose, hydroxpropyl-guar, hyaluronic acid, andhydroxypropyl cellulose) and the like. Additives in the formulation mayinclude, but are not limited to, sodium chloride, sodium bicarbonate,sorbic acid, methyl paraben, propyl paraben, chlorhexidine, castor oil,and sodium perborate.

Aqueous ophthalmic compositions (solutions or suspensions) generally donot contain physiologically or ophthalmically harmful constituents. Insome embodiments, purified or deionized water is used in thecomposition. The pH may be adjusted by adding any physiologically andophthalmically acceptable pH adjusting acids, bases or buffers to withinthe range of about 5.0 to 8.5. Ophthalmically acceptable examples ofacids include acetic, boric, citric, lactic, phosphoric, hydrochloric,and the like, and examples of bases include sodium hydroxide, sodiumphosphate, sodium borate, sodium citrate, sodium acetate, sodiumlactate, tromethamine, trishydroxymethylamino-methane, and the like.Salts and buffers include citrate/dextrose, sodium bicarbonate, ammoniumchloride and mixtures of the aforementioned acids and bases.

In some embodiments, the methods involve forming or supplying a depot ofthe therapeutic agent in contact with the external surface of the eye. Adepot refers to a source of therapeutic agent that is not rapidlyremoved by tears or other eye clearance mechanisms. This allows forcontinued, sustained high concentrations of therapeutic agent to bepresent in the fluid on the external surface of the eye by a singleapplication. Without wishing to be bound by any theory, it is believedthat absorption and penetration may be dependent on both the dissolveddrug concentration and the contact duration of the external tissue withthe drug containing fluid. As the drug is removed by clearance of theocular fluid and/or absorption into the eye tissue, more drug isprovided, e.g. dissolved, into the replenished ocular fluid from thedepot. Accordingly, the use of a depot may more easily facilitateloading of the ocular tissue for more insoluble therapeutic agents. Insome embodiments, the depot can remain for up to eight hours or more. Insome embodiments, the ophthalmic depot forms includes, but is notlimited to, aqueous polymeric suspensions, ointments, and solid inserts.

In some embodiments, the ophthalmic composition is an ointment or gel.In some embodiment, the ophthalmic composition is an oil-based deliveryvehicle. In some embodiments, the composition comprises a petroleum orlanolin base to which is added the active ingredient, usually as 0.1 to2%, and excipients. Common bases may include, but are not limited to,mineral oil, petrolatum and combinations thereof. In some embodiments,the ointment is applied as a ribbon onto the lower eyelid.

In some embodiment, the ophthalmic composition is an ophthalmic insert.In some embodiments, the ophthalmic insert is biologically inert, soft,bio-erodible, viscoelastic, stable to sterilization after exposure totherapeutic agents, resistant to infections from air borne bacteria,bio-erodible, biocompatible, and/or viscoelastic. In some embodiments,the insert comprises an ophthalmically acceptable matrix, e.g., apolymer matrix. The matrix is typically a polymer and the therapeuticagent is generally dispersed therein or bonded to the polymer matrix. Insome embodiments, the therapeutic agent may be slowly released from thematrix through dissolution or hydrolysis of the covalent bond. In someembodiments, the polymer is bioerodible (soluble) and the dissolutionrate thereof can control the release rate of the therapeutic agentdispersed therein. In another form, the polymer matrix is abiodegradable polymer that breaks down such as by hydrolysis to therebyrelease the therapeutic agent bonded thereto or dispersed therein. Infurther embodiments, the matrix and therapeutic agent can be surroundedwith an additional polymeric coating to further control release. In someembodiments, the insert comprises a biodegradable polymer such aspolycaprolactone (PCL), an ethylene/vinyl acetate copolymer (EVA),polyalkyl cyanoacrylate, polyurethane, a nylon, orpoly(dl-lactide-co-glycolide) (PLGA), or a copolymer of any of these. Insome embodiments, the therapeutic agent is dispersed into the matrixmaterial or dispersed amongst the monomer composition used to make thematrix material prior to polymerization. In some embodiments, the amountof therapeutic agent is from about 0.1 to about 50%, or from about 2 toabout 20%. In further embodiments, the biodegradable or bioerodiblepolymer matrix is used so that the spent insert does not have to beremoved. As the biodegradable or bioerodible polymer is degraded ordissolved, the therapeutic agent is released.

In further embodiments, the ophthalmic insert comprises a polymer,including, but are not limited to, those described in Wagh, et al.,“Polymers used in ocular dosage form and drug delivery systems”, AsianJ. Pharm., pages 12-17 (January 2008), which is incorporated herein byreference in its entirety. In some embodiments, the insert comprises apolymer selected from polyvinylpyrrolidone (PVP), an acrylate ormethacrylate polymer or copolymer (e.g., Eudragit® family of polymersfrom Rohm or Degussa), hydroxymethyl cellulose, polyacrylic acid,poly(amidoamine) dendrimers, poly(dimethyl siloxane), polyethyleneoxide, poly(lactide-co-glycolide), poly(2-hydroxyethylmethacrylate),poly(vinyl alcohol), or poly(propylene fumarate). In some embodiments,the insert comprises Gelfoam® R. In some embodiments, the insert is apolyacrylic acid of 450 kDa-cysteine conjugate.

In some embodiments, the ophthalmic composition is a ophthalmic film.Polymers suitable for such films include, but are not limited to, thosedescribed in Wagh, et al. (ibid), In some embodiments, the film is asoft-contact lens, such as ones made from copolymers ofN,N-diethylacrylamide and methacrylic acid crosslinked withethyleneglycol dimethacrylate.

In some embodiments, the ophthalmic composition comprises microspheresor nanoparticles. In some embodiment, the microspheres comprise gelatin.In some embodiments, the microspheres are injected to the posteriorsegment of the eye, in the chroroidal space, in the sclera,intravitreally or sub-retinally. In some embodiments, the microspheresor nanoparticles comprises a polymer including, but not limited to,those described in Wagh, et al. (ibid), which is incorporated herein byreference in its entirety. In some embodiments, the polymer is chitosan,a polycarboxylic acid such as polyacrylic acid, albumin particles,hyaluronic acid esters, polyitaconic acid, poly(butyl)cyanoacrylate,polycaprolactone, poly(isobutyl)caprolactone, poly(lacticacid-co-glycolic acid), or poly(lactic acid). In some embodiments, themicrospheres or nanoparticles comprise solid lipid particles.

In some embodiments, the ophthalmic composition comprises anion-exchange resin. In some embodiments, the ion-exchange resin is aninorganic zeolite or synthetic organic resin. In some embodiments, theion-exchange resin includes, but is not limited to, those described inWagh, et al. (ibid), which is incorporated herein by reference in itsentirety. In some embodiments, the ion-exchange resin is a partiallyneutralized polyacrylic acid.

In some embodiments, the ophthalmic composition is an aqueous polymericsuspension. In some embodiments, the therapeutic agent or a polymericsuspending agent is suspended in an aqueous medium. In some embodiments,the aqueous polymeric suspensions may be formulated so that they retainthe same or substantially the same viscosity in the eye that they hadprior to administration to the eye. In some embodiments, they may beformulated so that there is increased gelation upon contact with tearfluid.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to labeled version ofcertain compounds described herein (radio-labeled, fluorescent-labeled,etc.) that would be useful not only in imaging techniques but also inassays, both in vitro and in vivo, for localizing and quantitating JAKin tissue samples, including human, and for identifying JAK ligands byinhibition binding of a labeled compound. Accordingly, the presentinvention includes JAK assays that contain such labeled compounds.

The present invention further includes isotopically-labeled compounds.An “isotopically” or “radio-labeled” compound is a compound describedherein where one or more atoms are replaced or substituted by an atomhaving an atomic mass or mass number different from the atomic mass ormass number typically found in nature (i.e., naturally occurring).Suitable radionuclides that may be incorporated in compounds describedherein include but are not limited to ²H (also written as D fordeuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N,¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵Iand ¹³¹I. The radionuclide that is incorporated in the instantradio-labeled compounds will depend on the specific application of thatradio-labeled compound. For example, for in vitro metalloproteaselabeling and competition assays, compounds that incorporate ³H, ¹⁴C,⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S or will generally be most useful. Forradio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Bror ⁷⁷Br will generally be most useful.

It is understood that a “radio-labeled” or “labeled compound” is acompound that has incorporated at least one radionuclide. In someembodiments the radionuclide is selected from the group consisting of³H, ¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br.

The present invention can further include synthetic methods forincorporating radio-isotopes into compounds described herein. Syntheticmethods for incorporating radio-isotopes into organic compounds are wellknown in the art, and a person of ordinary skill in the art will readilyrecognize the methods applicable for the compounds described herein.

A labeled compound of the invention can be used in a screening assay toidentify/evaluate compounds. For example, a newly synthesized oridentified compound (i.e., test compound) which is labeled can beevaluated for its ability to bind a JAK by monitoring its concentrationvariation when contacting with the JAK, through tracking of thelabeling. For example, a test compound (labeled) can be evaluated forits ability to reduce binding of another compound which is known to bindto a JAK (i.e., standard compound). Accordingly, the ability of a testcompound to compete with the standard compound for binding to the JAKdirectly correlates to its binding affinity. Conversely, in some otherscreening assays, the standard compound is labeled and test compoundsare unlabeled. Accordingly, the concentration of the labeled standardcompound is monitored in order to evaluate the competition between thestandard compound and the test compound, and the relative bindingaffinity of the test compound is thus ascertained.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of JAK-associated diseases ordisorders, such as cancer, which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of an active compound. Such kits can further include,if desired, one or more of various conventional pharmaceutical kitcomponents, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES Example 1(R)-3-cyclopentyl-3-[4-(2-hydroxy-5-oxo-6,7-dihydro-5,1-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

Step 1.(R)-3-cyclopentyl-3-[4-(2-methoxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

4-Chloro-2-methoxy-7H-pyrrolo[2,3-d]pyrimidine (0.4 g, 2.18 mmol,Toronto Research Chemicals) and(R)-3-cyclopentyl-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]propanenitrile(0.824 g, 2.61 mmol, prepared as described in Org. Lett., 2009, 11(9),1999-2002.) were dissolved in 1,4-dioxane (4 mL) and potassium carbonate(0.903 g, 6.54 mmol) in water (2 mL) was added. The mixture was degassedand tetrakis(triphenylphosphine)palladium(0) (0.126 g, 0.109 mmol) wasadded. The reaction mixture was heated to 100° C. for 16 h. The reactionmixture was partitioned between water and ethyl acetate. The aqueouslayer was extracted with ethyl acetate three times. The combinedextracts were dried over sodium sulfate, decanted and concentrated.Flash column chromatography, eluting with a gradient from 0-10% MeOH inmethylene chloride was used to purify the product (670 mg, 91%). ¹H NMR(300 MHz, CD₃OD): δ 8.59 (s, 1H), 8.35 (s, 1H), 7.24 (d, 1H), 6.81 (d,1H), 4.47 (dt, 1H), 4.04 (s, 3H), 3.21 (dd, 1H), 3.10 (dd, 1H),2.62-2.44 (m, 1H), 2.02-1.86 (m, 1H), 1.81-1.20 (m, 7H); LCMS (M+H)⁺:337.0.

Step 2.(R)-3-cyclopentyl-3-[4-(5-iodo-2-methoxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

To a solution of3-cyclopentyl-3-[4-(2-methoxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(0.532 g, 1.58 mmol) in tetrahydrofuran (20 mL) was addedN-iodosuccinimide (0.36 g, 1.6 mmol). The reaction was stirred for 30min and the solvent was removed in vacuo. The residue was purified byflash column chromatography, eluting with a gradient from 0-65% ethylacetate in hexanes to afford a yellow solid (250 mg, 34%). ¹H NMR (300MHz, CDCl₃): δ 10.31 (br s, 1H), 8.27 (s, 1H), 8.23 (s, 1H), 7.29 (s,1H), 4.34-4.21 (m, 1H), 4.06 (s, 3H), 3.14 (dd, 1H), 3.03-2.90 (m, 1H),2.66-2.49 (m, 1H), 2.02-1.17 (m, 8H); LCMS (M+H)⁺: 463.0.

Step 3.(R)-4-[1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl]-2-methoxy-7H-pyrrolo[2,3-d]pyrimidin-5-ylacetate

A solution of3-cyclopentyl-3-[4-(5-iodo-2-methoxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(0.25 g, 0.54 mmol) in acetic acid (3 mL) was treated with silveracetate (0.27 g, 1.6 mmol) and heated to 70° C. for 16 h. The mixturewas filtered, rinsed with MeCN, water was added to the filtrate and thismixture was stirred for 20 min, Solid sodium chloride was added to thissolution. The product was obtained by extraction of this aqueous mixturewith three portions of ethyl acetate. The combined extracts were driedover sodium sulfate, decanted and concentrated. A portion of the productwas used in the hydrolysis step (Step 4) without further purification.¹H NMR (300 MHz, CDCl₃): δ 9.87 (br s, 1H), 8.39 (s, 1H), 8.37 (s, 1H),7.33 (s, 1H), 4.22 (dt, 1H), 4.06 (s, 3H), 3.14 (dd, 1H), 2.94 (dd, 1H),2.64-2.47 (m, 1H), 2.36 (s, 3H), 2.03-1.86 (m, 1H), 1.79-1.12 (m, 7H);LCMS (M+H)⁺: 395.1.

Step 4.(R)-3-cyclopentyl-3-[4-(2-hydroxy-5-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanamide

4 M of HBr in acetic acid (2 mL, 8 mmol) was added to4-[1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl]-2-methoxy-7H-pyrrolo[2,3-d]pyrimidin-5-ylacetate (0.050 g, 0.13 mmol) and the reaction was stirred for 1 h.Volatiles were removed in vacuo. The residue was reconstituted andpreparative HPLC-MS (eluting with a gradient of MeCN/H₂O containing0.15% NH₄OH) was used to afford purified product (12 mg, 26%). ¹H NMR(500 MHz, d₆-DMSO): δ 9.20 (s, 1H), 8.69 (s, 1H), 7.34 (s, 1H), 6.75 (s,1H), 4.49 (dt, 1H), 3.89 (s, 2H), 2.80 (dd, 1H), 2.64 (dd, 1H),2.36-2.26 (m, 1H), 1.83-1.74 (m, 1H), 1.63-1.36 (m, 4H), 1.32-1.20 (m,2H), 1.15-1.05 (m, 1H); LCMS (M+H)⁺: 357.0.

Step 5.(R)-3-cyclopentyl-3-[4-(2-hydroxy-5-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

To a solution of3-cyclopentyl-3-[4-(2-hydroxy-5-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanamide(0.006 g, 0.02 mmol) in methylene chloride (0.5 mL) containingtriethylamine (20 TL, 0.2 mmol) was added trichloroacetyl chloride (20TL, 0.2 mmol). When the reaction was complete, preparative HPLC-MS(MeCN/H₂O containing 0.15% NH₄OH) was used to afford purified product (3mg, 52%). ¹H NMR (400 MHz, d₆-DMSO): δ 11.39 (br s, 1H), 9.37 (s, 1H),8.77 (s, 1H), 8.63 (s, 1H), 4.68-4.59 (m, 1H), 3.94 (s, 2H), 3.19-3.15(m, 2H), 2.42-2.30 (m, 1H), 1.86-1.75 (m, 1H), 1.69-1.20 (m, 6H),1.18-1.05 (m, 1H); LCMS (M+H)⁺: 339.1.

Example 2 (3R)- and(3S)-3-[(1R,2R)-2-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand (3R)- and(3S)-3-[(1S,2S)-2-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

Step 1. (1S,2R)-ethyl2-(tert-butyldimethylsiloxy)cyclopentanecarboxylate and (1R,2S)-ethyl2-(tert-butyldimethylsilyloxy)cyclopentanecarboxylate

To a solution of tert-butyldimethylsilyl chloride (0.524 g, 3.48 mmol)and 1H-Imidazole (0.473 g, 6.95 mmol) in N,N-dimethylformamide (15 mL)was added ethyl cis-2-hydroxy-1-cyclopentanecarboxylate (racemic, Acros)(0.50 g, 0.0032 mol). The reaction was stirred for 16 h. Furtherimidazole (0.40 g, 5.8 mmol) and tert-butyldimethylsilyl chloride (0.50g, 3.3 mmol) were added portion wise and the reaction stirred for afurther 24 h. The product was extracted with hexane. The extracts werewashed with water, dried over sodium sulfate, filtered and evaporated toafford racemic TBS-protected hydroxyester (0.9 g) which were usedwithout further purification. ¹H NMR (400 MHz, CDCl₃): δ 4.46 (ddd, 1H),4.19 (dq, 1H), 4.01 (dq, 1H), 2.72 (dt, 1H), 2.22-2.11 (m, 1H),1.96-1.49 (m, 5H), 1.26 (t, 3H), 0.84 (s, 9H), 0.03 (s, 3H), 0.01 (s,3H).

Step 2. (1S,2R)-2-(tert-butyldimethylsilyloxy)cyclopentanecarbaldehydeand (1R,2S)-2-(tert-butyldimethylsilyloxy)cyclopentanecarbaldehyde

To a solution of (1S,2R)-ethyl2-(tert-butyldimethylsilyloxy)cyclopentanecarboxylate and (1R,2S)-ethyl2-(tert-butyldimethylsilyloxy)cyclopentanecarboxylate from Step 1 (0.86g, 3.2 mmol) in hexanes (40 mL) at −78° C. was added drop wise asolution of 1.0 M of diisobutylaluminum hydride in toluene (3.5 mL, 3.5mmol). The reaction mixture was stirred for 1 h at −78° C. and wasquenched this temperature by the drop wise addition of methanol (2 mL).Cooling was discontinued and the mixture was allowed to reach ambienttemperature. An aqueous solution of Rochelle's salt was added. Thebiphasic mixture was stirred vigorously for 2 h and the resulting layerswere separated. The aqueous layer was extracted once further withhexanes, then with three portions of ethyl acetate. The combinedextracts were washed with brine, dried over sodium sulfate, decanted andconcentrated to afford the racemic aldehyde product, used withoutfurther purification (0.7 g, 97%). ¹H NMR (400 MHz, CDCl₃): δ 9.74 (d,1H), 4.62 (ddd, 1H), 2.68-2.61 (m, 1H), 2.22-2.11 (m, 1H), 1.95-1.83 (m,1H), 1.80-1.57 (m, 4H), 0.85 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H).

Step 3. (E)- and(Z)-3-((1R,2R)-2-(tert-butyldimethylsilyloxy}cyclopentyl)acrylonitrileand (E)- and(Z)-3-((1S,2S)-2-(tert-butyldimethylsilyloxy}cyclopentyl)acrylonitrile

To a solution of(1S,2R)-2-(tert-butyldimethylsilyloxy)cyclopentanecarbaldehyde and(1R,2S)-2-(tert-butyldimethylsilyloxy)cyclopentanecarbaldehyde (0.36 g,1.6 mmol, from Step 2) in toluene (9 mL) was added(triphenylphosphoranylidene)acetonitrile (0.475 g, 1.58 mmol) and thereaction was heated to 80° C. for 2 h. The reaction was cooled to roomtemperature and water was added. The product was extracted with threeportions of ethyl ether. The extracts were washed with brine, dried oversodium sulfate, decanted and concentrated to provide the racemic mixtureof E- and Z-olefin isomers which was used without further purification.¹H NMR (400 MHz, CDCl₃): δ 6.82 (dd, 1H, trans olefin), 6.63 (dd, 1H,cis olefin), 5.307 (dd, 1H, trans olefin), 5.305 (dd, 1H, cis olefin),4.24 (ddd, 1H), 4.20 (ddd, 1H), 2.96-2.86 (m, 1H), 2.53-2.43 (m, 1H),1.95-1.56 (m, 12H), 0.87 (s, 9H), 0.86 (s, 9H), 0.04-0.01 (singlets,together 12H).

Step 4. (3R)- and(3S)-3-[(1R,2R)-2-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]-pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand (3R)- and(3S)-3-[(1S,2S)-2-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

To a solution of (E)- and(Z)-3-((1R,2R)-2-(tert-butyldimethylsilyloxy)cyclopentyl)acrylonitrileand (E)- and(Z)-3-((1S,2S)-2-(tert-butyldimethylsilyloxy)cyclopentyl)acrylonitrile(0.40 g, 1.6 mmol, crude product from Step 3) in acetonitrile (20 mL)was added4-(1H-pyrazol-4-yl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine(0.50 g, 1.6 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.24 mL,1.6 mmol). The reaction was stirred for 2 h at room temperature, andfurther DBU (0.24 mL, 1.6 mmol) was added. The reaction was stirred for3 days and was concentrated. Flash column chromatography (eluting with agradient from 10-40% ethyl acetate/hexanes) was used to purify productwhich was then treated with 20% TFA in DCM for 3 h, evaporated, andtreated with excess ethylenediamine in methanol solution overnight. Whenremoval of SEM protecting group was complete, any remaining TBSprotecting group was removed by stirring with EtOH/H₂O/c.HC1 for 3 hours(10:4:3 volume ratio). The fully deprotected products were purified bypreparative HPLC-MS (0.15% NH₄OH in a gradient of MeCN/H₂O). Allfractions of M+H=323 were pooled and evaporated (approximately 80 mg).The products were subjected to a series of chiral chromatographicpurifications as follows: Chiral Technologies Chiralcel OJ-H (3×25 cm, 5Tm) eluting with 20% EtOH/80% Hexanes at a flow rate of 25 mL/min toafforded Peak 1 (19 mg), the following minor peak not collected; Peak 2(60 mg), Peak 3 (6 mg). Peak 2 was a mixture which was then furtherseparated using Chiral Technologies Chiralpak IA (2×25 cm, 5 Tm) elutingwith a gradient of 70% EtOH/30% Hexanes at a flow rate of 8 mL/min intothree components. These were labeled Peak 2-1 (run 2, peak 1, 32 mg)which was a mixture of products, Peak 2-2 (6.5 mg) and Peak 2-3 (13.7mg). Peak 2-1 was further separated into three components using ChiralTechnologies Chiralpak IA (2×25 cm, 5 Tm) eluting with a gradient of 25%EtOH/75% Hexanes at a flow rate of 12 mL/min. The isolated products werelabeled Peak 2-1-1 (10.5 mg); 2-1-2 (13 mg); and 2-1-3 (2.3 mg).

Peak 1: ¹H NMR (500 MHz, CD₃OD): δ 8.65 (s, 1H), 8.62 (s, 1H), 8.37 (s,1H), 7.49 (d, 1H), 6.95 (d, 1H), 4.71 (ddd, 1H), 4.28 (br t, 1H), 3.26(dd, 1H), 3.21 (dd, 1H), 2.53-2.45 (m, 1H), 1.98-1.73 (m, 3H), 1.62-1.47(m, 2H), 1.38-1.29 (m, 1H); LCMS (M+H)⁺: 323.

Peak 2-1-1: ¹H NMR (300 MHz, CD₃OD): δ 8.64 (s, 1H), 8.59 (s, 1H), 8.38(s, 1H), 7.49 (d, 1H), 6.94 (d, 1H), 4.90-4.78 (m, 1H), 3.64 (br t, 1H),3.21 (dd, 1H), 3.07 (dd, 1H), 2.55-2.40 (m, 1H), 2.01-1.58 (m, 6H); LCMS(M+H)⁺: 323.

Peak 2-1-2: ¹H NMR (500 MHz, CD₃OD): δ 8.65 (s, 1H), 8.62 (s, 1H), 8.37(s, 1H), 7.49 (d, 1H), 6.95 (d, 1H), 4.71 (ddd, 1H), 4.28 (br t, 1H),3.26 (dd, 1H), 3.21 (dd, 1H), 2.53-2.45 (m, 1H), 1.98-1.73 (m, 3H),1.62-1.48 (m, 2H), 1.38-1.26 (m, 1H); LCMS (M+H)⁺: 323.

Peak 2-3: ¹H NMR (300 MHz, CD₃OD): δ 8.64 (s, 1H), 8.59 (s, 1H), 8.38(s, 1H), 7.48 (d, 1H), 6.93 (d, 1H), 4.90-4.78 (m, 1H), 3.64 (br t, 1H),3.21 (dd, 1H), 3.07 (dd, 1H), 2.55-2.40 (m, 1H), 2.01-1.58 (m, 6H); LCMS(M+H)⁺: 323.

Example 3 Racemic3-(1-Hydroxycyclopentyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetate salt

Step 1. 1-[(trimethylsilyl)oxy]cyclopentanecarbaldehyde

The reaction was carried out in a procedure similar to that described inTetrahedron, 50(9), 2821-30; 1994: To a solution of1-[(trimethylsilyl)oxy]cyclopentanecarbonitrile (2.25 g, 12.3 mmol,prepared as described in Organometallics, 3(11), 1660-5; 1984) intoluene (18 mL) at −45° C. was added dropwise 1.0 M ofdiisobutylaluminum hydride in hexane (17.2 mL, 17.2 mmol). The solutionwas then allowed to warm to 0° C. and stir for 1 h at this temperature.The reaction mixture was poured into a mixture of diethyl ether (25 mL)and ammonium chloride (25 mL, saturated). To the resulting mixture, at15° C., was added a dilute solution of sulfuric acid (prepared bydiluting 1.53 mL of concentrated H₂SO₄ with 50 mL water). The solutionwas then stirred at a temperature of 5° C. overnight. The mixture wasextracted with three portions of diethyl ether, the combined extractswere washed with brine, dried over sodium sulfate, decanted andconcentrated to afford product (0.84 g, 36%), which was used withoutfurther purification in Step 2. ¹H NMR (300 MHz, CDCl₃): δ 9.47 (s, 1H),1.88-1.46 (m, 8H), 0.00 (s, 9H).

Step 2. (2E)- and(2Z)-3-{1-[(trimethylsilyl)oxy]cyclopentyl}acrylonitrile

Diethyl cyanomethylphosphonate (0.912 mL, 5.64 mmol) was added dropwiseto a suspension of sodium hydride (0.198 g, 4.96 mmol) intetrahydrofuran (10 mL) at 0° C. Following addition, the reaction waswarmed to room temperature and stirred for 45 minutes. The mixture wasrecooled to 0° C. and 1-[(trimethylsilyl)oxy]cyclopentanecarbaldehyde(0.84 g, 4.5 mmol) in tetrahydrofuran (20 mL) was introduced. Thereaction was allowed to warm to room temperature and stir for 2 hours.Water and ethyl acetate were added into the reaction and the layersseparated. The aqueous layer was extracted with two further portions ofethyl acetate. The combined organic extracts were washed with brine,dried over sodium sulfate, decanted and concentrated to afford productas a mixture of olefin isomers, used without further purification inStep 3. ¹H NMR (300 MHz, CDCl₃): δ 6.80 (d, 1H, trans/major product),6.53 (d, 1H, cis/minor product), 5.52 (d, 1H, trans), 5.28 (d, 1H, cis),2.06-0.76 (m, 16H total for both isomers), 0.18 (s, 9H, minor product),0.13 (s, 9H, major product).

Step 3. racemic3-(1-hydroxycyclopentyl)-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile

To a suspension of (2E)- and(2Z)-3-{1-[(trimethylsilyl)oxy]cyclopentyl}acrylonitrile (0.94 g, 4.5mmol) and4-(1H-pyrazol-4-yl)-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine(1.4 g, 4.5 mmol) in acetonitrile (20 mL) was added1,8-diazabicyclo[5.4.0]undec-7-ene (0.67 mL, 4.5 mmol). The reaction wasallowed to stir at room temperature for 6 days. The acetonitrile wasevaporated. The desired unprotected alcohol product was isolated fromthe mixture of unprotected alcohol and TMS-protected alcohol productsusing flash column chromatography, eluting with a gradient from 0-80%ethyl acetate in hexanes (890 mg, 44%). ¹H NMR (300 MHz, CDCl₃): δ 8.87(s, 1H), 8.56 (br s, 1H), 8.35 (s, 1H), 7.46 (d, 1H), 6.84 (d, 1H), 5.69(s, 2H), 4.40 (dd, 1H), 4.03 (s, 1H), 3.55 (dd, 2H), 3.46 (dd, 1H), 2.97(dd, 1H), 2.04-1.27 (m, 8H), 0.93 (dd, 2H), −0.05 (s, 9H); LCMS (M+H)⁺:453.1.

Step 4. racemic3-(1-hydroxycyclopentyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetate salt

A solution of racemic3-(1-hydroxycyclopentyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(0.100 g, 0.221 mmol) in methylene chloride (4 mL) and trifluoroaceticacid (1 mL) was stirred for 2 hours and the solvents were evaporated.The residue was stirred with ethylenediamine (0.1 mL, 2 mmol) inmethanol (4.5 mL) for 1 hour and evaporated. The crude product wasreconstituted in methanol and purified by two successive chromatographicsteps using preparative HPLC-MS (eluting with a gradient ofacetonitrile/H₂O containing 0.15% NH₄OH for the first run, followed by agradient of acetonitrile/H₂O containing 0.1% TFA for the second run) toafford the racemic product as the trifluoroacetate salt. ¹H NMR (400MHz, d₆-dmso): δ 12.72 (br s, 1H), 8.94 (s, 1H), 8.86 (s, 1H), 8.49 (s,1H), 7.82 (s, 1H), 7.21 (s, 1H), 4.80 (dd, 1H), 3.51 (dd, 1H), 3.22 (dd,1H), 1.79-1.42 (m, 7H), 1.25-1.15 (m, 1H); LCMS (M+H)⁺: 323.1.

Example A

Metabolites 1-39 were isolated from human, rat or dog urine, plasma, orfeces after administration of Compound I in connection withpharmacokinetic and toxicokinetic studies. The identity of themetabolites were determined after isolation of the metabolite using HPLCmethods. The method used and the corresponding retention times of themetabolites are presented in Table 2. Tandem MS/MS analysis and higherorder MS^(n) experiments were conducted as required to elucidatestructural information.

Compound I demonstrates a protonated molecular ion at m/z 307, and aproduct ion spectrum showing signals at m/z 266, 186, and 159. Thefragment ion at m/z 266 is consistent with the loss of the carbonitrilemoiety. The diagnostic fragment ion at m/z 186 is indicative of the lossof the intact cyclopentylpropanenitrile moiety. The fragment ion at m/z159 indicates the loss of the cyclopentylpropanenitrile in conjunctionwith cleavage through the pyrazole ring of Compound I.

Metabolite compounds 1, 2, 27, and 29 were observed primarily in urinefrom human subjects, with trace levels of compounds 27 and 29 observedin plasma. Full scan mass spectrometry demonstrated a protonatedmolecular ion at m/z 339, consistent with bis-hydroxylation of CompoundI. Product ion fragmentation of these m/z 339 ions yields virtuallyidentical spectra for these metabolites, with ions observed at m/z 321,186, 159 and 154. The ion at m/z 321 is consistent with a water loss andsuggests that at least one hydroxylation may be on the cyclopentyl ring.The ions at m/z 186 and m/z 159 are consistent with an intactpyrazole-pyrrolopyrimidine. The ion at m/z 154 is consistent with aneutral loss of the entire unmodified pyrazole/pyrrolopyrimidine moiety.Minor fragment ions at m/z 298 and m/z 280 suggest the loss of theelements of acetonitrile, with and without the facile water loss,further restricting the location of the hydroxylations to thecyclopentyl moiety. This is further supported by the ion at m/z 237,which is consistent with the loss of the modified cyclopentyl, leavingthe rest of the molecule unmodified.

Compound 31 was found in urine from human subjects. Full scan massspectrometry demonstrated a protonated molecular ion at m/z 339,consistent with the addition of 32 amu to Compound I. Product ionfragmentation of the m/z 339 ion yields fragment ions at m/z 311, 218,and 191. The primary fragment at m/z 218 which is consistent with theaddition of 32 amu to the pyrazole-pyrrolopyrimidine moiety. Thefragment ion at m/z 191 is consistent with a loss of CHN from thepyrazole moiety of the putative bis hydroxylatedpyrazole-pyrrolopyrimidine, restricting the location of themodification(s) to the pyrrolopyrimidine. The fragment ion at m/z 311likely arises from initial cleavage of the amide bond, followed by theloss of CO from the pyrrolidinone. Structural assignment of Compound 31using mass spectrometry alone led to an ambiguous result, thereforeCompound 31 was isolated from human urine and analyzed by ¹H and ¹³CNMR. The structure of 31 is identified as an amide-alcohol metabolite ofthe saturated pyrrolopyrimidine moiety of Compound I. The proton NMRspectrum of 31 has a singlet at δ 3.56 with an intensity of 2H. Thissinglet has a long range correlation to a carbon at δ 177.9, consistentwith an amide. In addition, a nuclear Overhauser enhancement (nOe)occurs between H3 and H9.

Compound 32 was found in plasma and urine from human subjects. Full scanmass spectrometry demonstrated a protonated molecular ion at m/z 339,consistent with the addition of 32 amu to Compound I. Product ionfragmentation of the m/z 339 ion yields fragment ions at m/z 218, and191. The primary fragment at m/z 218 which is consistent with theaddition of 32 amu to the pyrazole-pyrrolopyrimidine moiety. Thefragment ion at m/z 191 is consistent with a loss of CHN from thepyrazole moiety of the putative bis hydroxylatedpyrazole-pyrrolopyrimidine, restricting the location of themodification(s) to the pyrrolopyrimidine. Structural assignment ofCompound 32 using mass spectrometry alone led to an ambiguous result,therefore Compound 32 was isolated from human urine and analyzed by ¹Hand ¹³C NMR. The structure of Compound 32 is identified as aketo-alcohol metabolite of the saturated pyrrolopyrimidine moiety ofCompound I. The proton NMR spectrum of 32 has a singlet at δ 3.82 whichhas an intensity of 2H and shows a long range correlation to at carbonat δ 191.5, consistent with a ketone. There was no nuclear Overhauserenhancement (nOe) observed from H2 to any other proton.

Compound 40 was observed in plasma and urine from human subjects. Fullscan mass spectrometry demonstrated a protonated molecular ion at m/z341, consistent with the addition of 34 amu to Compound I. Product ionfragmentation of the m/z 341 ion yields fragment ions at m/z 323, 220,202, and 175. The fragment ion at m/z 323 arises from the loss of waterfrom the pyrrolidine diol. Fragment ions at m/z 220 and 202 areconsistent with a doubly hydroxylated saturated pyrrolopyrimidine, withand without the observed facile water loss. The ion observed at m/z 175is consistent with loss of CHN from the pyrazole moiety of the doublyhydroxylated saturated pyrazole pyrrolopyrimidine following the facilewater loss. Compound 40 is identified as3-cyclopentyl-3-(4-(5,6-dihydroxy-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile.

Conjugates of singly hydroxylated metabolites of Compound I having anobserved m/z of 499 were observed in urine from human, with two of theseven conjugates also observed in trace amounts in human plasma. Initialproduct ion fragmentation yields virtually identical spectra for six ofthese metabolites, with an observed ion at m/z 323, consistent with theloss of the glucuronide conjugate. The product ion fragmentation (MS³)of these m/z 323 fragment ions again demonstrated virtually identicalmass spectra, with fragment ions at m/z 305, 186, and 159. The fragmention found at m/z 305 is consistent with a loss of water (18 amu),suggesting hydroxylation and subsequent collision induced fragmentationon a saturated portion of the molecule, restricting the site ofmodification to the cyclopentyl moiety. The fragment ions at m/z 186 andm/z 159 are consistent with an unmodified pyrazole-pyrrolopyrimidine.Additional minor fragment ions were too weak to assign them with anycertainty. The identity of these putative metabolites was confirmed byhydrolysis of the isolated glucuronide conjugates with β-Glucuronidaseto reveal the aglycone, which was then subject to analysis by HPLC-MSwhere the retention times and mass spectra of the liberated aglyconeswere matched with those of singly hydroxylated metabolite standards. Theaglycones of these glucuronide conjugate metabolites correspond to2-hydroxyl metabolites (Metabolite 9, Table 1) and 3-hydroxylmetabolites (Metabolite 8, Table 1). The aglycone of the seventhglucuronide conjugate metabolite, compound 42, corresponds to a singlyhydroxylated metabolite of the pyrazole-pyrrolopyrimidine moiety ofCompound I, which was not independently observed in plasma or urine fromhuman subjects. Full scan mass spectrometry demonstrated a protonatedmolecular ion at m/z 323, consistent with the addition of 16 amu toCompound I. Product ion fragmentation of the m/z 323 ion yields aprimary fragment at m/z 202 which is consistent with the addition of 16amu to the pyrazole-pyrrolopyrimidine moiety. The fragment ion at m/z175 is consistent with a loss of CHN from the pyrazole moiety of theputative hydroxylated pyrazole-pyrrolopyrimidine, restricting thelocation of the modification to the pyrrolopyrimidine. The structure ofmetabolite compound 42 is identified as a glucuronide conjugate of asingly hydroxylated pyrrolopyrimidine moiety of Compound I.

Metabolite compound 41 was found in urine from human subjects. Full scanmass spectrometry indicated a protonated molecular ion at m/z 583,consistent with glucuronide conjugation of unmodified Compound I.Product ion fragmentation of the m/z 583 ion yields a primary fragmentat m/z 307 which is consistent with the loss of an intact glucuronide.Minor fragment ions at m/z 186 and m/z 159 are consistent with thoseobserved for Compound I. MS³ fragmentation of the m/z 307 fragment ionalso revealed fragment ions at m/z 186 and m/z 159. Compound 41 isidentified as an N-linked glucuronide conjugate of Compound I.

TABLE 2 Re- Anal- tention Com- ysis time pound m/z method (min)Metabolite description 1 339 A4 4.3 Di-hydroxylation of cyclopentylmoiety 2 339 A4 4.8 Di-hydroxylation of cyclopentyl moiety 3 499 A3 4.86-(3-(1-(4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-2-cyanoethyl)cyclo- pentyloxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid 4 499 A3 5.3 O-glucuronidation ofcyclopentyl moiety 5 499 A3 5.8 O-glucuronidation of cyclopentyl moiety6 339 A4 5.9 Dihydroxylation 7 499 A3 6.3 O-glucuronidation ofcyclopentyl moiety 8 499 A3 6.8 6-(3-(1-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)- 2-cyanoethyl)cyclopentyloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid 9 339 A4 8.1Hydroxylation on cyclopentyl and pyrazole or pyrrolopyrimidine moieties10 339 A4 8.4 Hydroxylation on cyclopentyl and pyrazole orpyrrolopyrimidine moieties 11 355 A1 8.5 Tri-hydroxylation ofcyclopentylpropanenitrile moiety 12 355 A1 14.6 Tri-hydroxylation ofcyclopentylpropanenitrile moiety 13 499 A1/A3 15.3 O-glucuronidation ofcyclopentylpropanenitrile moiety 14 499 A1/A3 18.6 O-glucuronidation ofcyclopentylpropanenitrile moiety 15 499 A1/A3 25.3 O-glucuronidation ofcyclopentylpropanenitrile moiety 16 321 A1/A3 26.5 ketone oncyclopentylpropanenitrile moiety 17 339 A1 28 Hydroxylation oncyclopentylpropanenitrile and pyrrolidine moieties 18 323 A1/A3 35Hydroxylation of the cyclopentylpropanenitrile moiety 19 499 A1/A3 42.2O-glucuronidation of cyclopentylpropanenitrile moiety 20 355 A1 44.2Di-hydroxylation of cyclopentylpropanenitrile moiety and hydroxylationof pyrrolidine moiety 21 355 A1 51.4 Di-hydroxylation ofcyclopentylpropanenitrile moiety and hydroxylation of pyrrolidine moiety22 339 A1 52.2 Hydroxylation on cyclopentylpropanenitrile andpyrrolidine moieties 23 339 A1 55.8 Hydroxylation oncyclopentylpropanenitrile and pyrrolidine moieties 24 355 A1 4.7Dihydroxylation on cyclopentylpropanenitrile moiety and hydroxylation ofpyrrolidine moiety 25 339 A1 7.7 Di-hydroxylation oncyclopentylpropanenitrile moiety 26 339 A1 8.4 Di-hydroxylation oncyclopentylpropanenitrile moiety 27 339 A1 11.9 Di-hydroxylation oncyclopentylpropanenitrile moiety 28 323 A1/A3 12.4 Hydroxylation oncyclopentylpropanenitrile moiety 29 339 A1 12.7 Di-hydroxylation ofcyclopentylpropanenitrile moiety 30 515 A3 9.6 Di-hydroxylation ofcyclopentylpropanenitrile moiety and O-glucuronidation 31 339 A3 273-cyclopentyl-3-(4-(2,6-dioxo- 3,5,6,7-tetrahydro-2H-pyrrolo[2,3-d]pyrimidin- 4-yl)-1H-pyrazol-1-yl)propanenitrile 32 339 A329 3-cyclopentyl-3-(4-(2-hydroxy-5-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimi- din-4-yl)-1H-pyrazol-1-yl)propanenitrile 33 323 A1 55.8 Hydroxylation of pyrrolopyrimidinemoiety 34 499 A1/A3 37.5 O-glucuronidation of cyclopentylpropanenitrilemoiety 35 339 A6 5.1 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1,2- dihydroxycyclopentyl)propanenitrile 36323 — — 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile 37 323 A6 8.13-cyclopentyl-3-(4-(6-oxo-6,7- dihydro-5H-pyrrolo[2,3-d]-pyrimidin-4-yl)-1H- pyrazol-1-yl)propanenitrile 38 339 A6 5.43-(3-hydroxycyclopentyl)-3-(4-(6-oxo- 6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl)propanenitrile 39 371 A3 17.7quadrupole hydroxylation 40 341 A7 27.73-cyclopentyl-3-(4-(5,6-dihydroxy-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin- 4-yl)-1H-pyrazol-1-yl)propanenitrile41 483 A7 36.0 6-(4-(l-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]-pyrimidin-7-yl)-3,4,5-trihydroxytetra- hydro-2H-pyran-2-carboxylic acid42 499 A7 37.7 Hydroxylation of pyrrolopyrimidine moiety andO-glucuronidationMethod A1: Biotransformation of ¹⁴C-Compound I in Human, Dog, and MousePlasma, Urine, and Fecal SamplesSample Preparation for Metabolite Profiling:Plasma:

Plasma samples from all subjects were pooled by subject and/or by timepoint. The pooled plasma samples were prepared for HPLC-Radiometricanalysis as follows: Aliquots of the pooled plasma samples wereinitially extracted with approximately two equivalent (W/V) volumes of1% formic acid in HPLC-grade acetonitrile, followed by vigorousvortexing, and centrifugation to remove denatured plasma proteins. Thesupernatant was reserved and passed through a pre-conditioned WatersC-18 Solid phase extraction cartridge, and the filtrate retained. TheSPE cartridge was retained. The remaining plasma pellet was thenextracted three times with 1% formic acid in HPLC-gradeacetonitrile/water (90/10), with centrifugation after each extraction.All supernatants were reserved. Each formic acid/acetonitrile/waterextraction supernatant was used to elute the SPE cartridge. The SPEcartridge was then rinsed with 1% formic acid in methanol. All eluantswere retained, combined, and evaporated under a stream of nitrogen at30° C. The remainder of this extraction was reconstituted inapproximately 0.6 ml of 0.1% formic acid in water, vortexed andcentrifuged to remove particulate matter. The supernatant was thenanalyzed by HPLC-Flow Radiometry and HPLC-MS.

Urine:

Aliquots of approximately equal amounts of urine from each of thesubjects were pooled to represent appropriate time intervals post-dose.Prior to HPLC-Radiometric analysis, each pooled urine sample wascentrifuged at 4,000 rpm for 10 min to remove particulate matter, thenanalyzed directly.

Feces:

Aliquots of fecal homogenates from each subject were pooled to representappropriate time intervals post-dose. The pooled fecal homogenatesamples were prepared for HPLC-Radiometric analysis as follows:

Aliquots of approximately 5 grams of fecal homogenate were initiallyextracted with approximately two equivalent (W/V) volumes of Milliporewater, followed by centrifugation to remove solids. The supernatant wasreserved and passed through a pre-conditioned Waters C-18 Solid phaseextraction cartridge. The SPE cartridge was retained. The remainingfecal pellet was then extracted three times with 1% formic acid inHPLC-grade acetonitrile/water (90/10), with centrifugation after eachextraction. All supernatants were reserved. Each formicacid/acetonitrile/water extraction supernatant was used to elute the SPEcartridge. All eluants were retained, combined, and evaporated under astream of nitrogen at 30° C. The remainder of this extraction wasreconstituted in approximately 2.0 ml of 0.1% formic acid in water,vortexed and centrifuged to remove particulate matter. The supernatantwas then analyzed by HPLC-Flow Radiometry and HPLC-MSHPLC-Flow Radiometry Analytical Method:

All samples were analyzed by either HPLC-Flow radiometry or HPLC-MSusing analytical systems that were as identical as practicable. TheHPLC-Flow Radiometry system consisted of an Agilent HP1100 seriesQuaternary pump, Autosampler, and UV detector interfaced with a RaytestRamona-90 flow scintillation detector with a 500 μL liquid scintillationcell. Separation of Compound I and its metabolites was achieved with aWaters Symmetry C-18 HPLC column, 4.6×250 mm, 5 μm particle size HPLCcolumn. Mobile phase “A” consisted of 0.1% formic acid in HPLC-gradewater, with mobile phase “B” consisting of HPLC-grademethanol/acetonitrile (1:1). Elution of analytes was achieved throughuse of a linear increasing gradient of mobile phase “B”. Two differentgradient elution methods were employed at various times as needed.Chromatographic conditions for these two methods are outlined below inTables 3 and 4.

HPLC-Mass Spectrometry Analytical Method:

The HPLC-Flow Radiometry system consisted of an Agilent HP1100 seriesQuaternary pump, and Leap Technologies CTC-PAL Autosampler interfacedwith an Applied Biosystems API 4000 Qtrap operated in positive iondetection mode. Full scan (MS) and data dependent product ion scans(MS²) were employed in the characterization of metabolites of CompoundI. Select MRM transitions were also used to scan for and identifypreviously identified and characterized metabolites. Separation ofCompound I and its metabolites was achieved with a Waters Symmetry C-18HPLC column, 4.6×250 min, 5 μm particle size HPLC column. Mobile phase“A” consisted of 0.1% formic acid in HPLC-grade water, with mobile phase“B” consisting of HPLC-grade methanol/acetonitrile (1:1). Elution ofanalytes was achieved through use of a linear increasing gradient ofmobile phase “B”. Two different gradient elution methods were employedat various times as needed. Chromatographic conditions for these twomethods are outlined below in Tables 3 and 4.

TABLE 3 Chromatographic Conditions Time (min) % B Flow (μl/min) 0.0 101000 45.0 20 1000 50.0 50 1000 55.0 90 1000 57.0 90 1000 57.1 10 100065.0 10 1000

TABLE 4 Chromatographic Conditions Time (min) % B Flow (μl/min) 0.0 101000 45.0 20 1000 90.0 40 1000 92.0 90 1000 97.0 90 1000 97.1 10 1000102.0 10 1000Method A2: Biotransformation of Compound I: Isolation of MetabolitesIsolation of Metabolites from Human Urine:

Solid phase extraction using 20 cc Waters HLB SPE cartridges (1 gramsorbent) were used to concentrate metabolites of Compound I from pooledurine samples. The cartridges were conditioned first with 100% HPLCgrade methanol, then with Millipore water. The cartridge was thenimmediately loaded with up to 10 ml of raw unprocessed urine. Thecartridge was the sequentially eluted using several concentrations ofHPLC grade methanol in water (Table 5).

TABLE 5 Operation Solvent Composition Volume Condition 100% Methanol 10ml Condition 100% Millipore Water 10 ml Load Urine 7-10 ml Elute  5%Methanol in Water 10 ml Elute  25% Methanol in Water 10 ml Elute  50%Methanol in Water 10 ml Elute  75% Methanol in Water 10 ml Elute 100%Methanol in Water 10 ml

Aliquots of the initial urine, the last load volume, and everywash/elution volume were analyzed by LC-MS for the presence of CompoundI and metabolites of interest. There were no significant amounts ofeither Compound I, or its metabolites in any of the load volumes, orwashes/elutions up to 25% methanol in Millipore water. No significantamounts of drug or metabolites were found in the 100% methanol elutionaliquots. Elution volumes from the 50% and 75% methanol elutions wereeach reduced in volume using the Genevac centrifugal evaporator andreconstituted to a final volume of ˜2 mL before being serially injectedonto the LC-MS for fraction collection of the metabolites of interest.

Analytical Conditions:

Samples were analyzed by LC-MS using a Finnigan LCQ Deca XP Plusinterfaced with a Shimadzu Binary HPLC stack consisting of a Sil-HTCAutosampler and system controller, and two Sil 10ADVp high pressure LCpumps. The mass spec was operated in positive ion detection mode, usingdata dependent scanning to yield. MS and data-dependent MS² data. AShimadzu UV detector, Sil SPD10 AVp was used at a detection wavelengthof 254 nm to monitor the HPLC eluant in concert with the massspectrometer.

Mobile phase “A” consisted of 5 mM ammonium formate which was pHadjusted to pH 3.2 with formic acid (approximately 0.1% by volume).Mobile phase “B” consisted of 90% acetonitrile/10% methanol. The HPLCcolumn used was a Zobax XDB C-18, 3.0 mm×150 mm, 5.0 Tm particle size.Elution of analyte was through use of a linearly increasing gradient ofmobile phase “B”. Chromatographic conditions for the analytical scaleanalysis are outlined in Table 6 below.

TABLE 6 Chromatographic Conditions Time (min) % B Flow (Tl/min) 0.0 10300 1.0 10 300 45.0 40 300 50.0 90 300 54.0 90 300 54.1 10 300 60.0 10300Semi-PREP Analytical Conditions.

Samples were analyzed by LC-MS as above with the following changes:

The HPLC column used was a Phenomenex Polar RP 10 mm×150 mm 5 Tmparticle size.

The initial fractionation was carried out using neutral ammonium acetateas the “A” phase, with a second cleanup step of individual collectedpeaks carried out using 0.025% formic acid in water as the “A” phase.Mobile phase “B” consisted of 90% acetonitrile/10% methanol.

Initial elution of analytes and automated fraction collection wasthrough use of a linearly increasing gradient of mobile phase “B”.Chromatographic conditions for the initial semi preparative analysis areoutlined in Table 7 below, but % B was optimized as needed for eachindividual metabolite in the second cleanup step.

TABLE 7 Semi-Prep Chromatographic Conditions Time (min) % B Flow(ml/min) 0.0 7 1.75 1.0 25* 1.75 24.0 35* 1.75 24.1 90  1.75 28.0 90 1.75 28.1 7 1.75 35.0 7 1.75 *Optimized for each individual metabolitein second fractionation step

The eluant stream of the semi-prep analyses were split using a PEEK teeand tubing, with ˜1.65 ml/min of the eluant stream being utilized forfraction collection, and the remainder going to the mass spectrometer.The eluant stream composition was monitored by MS and selected MS-MSexperiments. Fractions containing the analyte of interest were collectedautomatically by utilizing the divert valve in concert with a fractioncollector.

Method A3: Biotransformation of Compound I in Human and Rat Plasma andUrine Samples

Sample Preparation:

Plasma and urine samples from animal and human subjects which had beenorally dosed with Compound I were obtained following single dose and/ormultiple dose studies. Residual plasma samples for each subject werepooled by time-weighted-average pooling. Aliquots of the pooled plasmasamples (150 μL) were precipitated with two volumes of acetonitrile,vortex mixed and then centrifuged. The supernatants were removed andused for analysis by LC-MS. For human, the urine samples from eachindividual in the study were pooled to represent 0-12 hours on Day 1(single dose study) and Day 10 (multiple dose study) of the clinicalstudy. Urine samples were centrifuged at ˜15000×g for 10 minutes beforeanalysis and injected directly.

Sample Analysis:

Samples were assayed using electrospray ionization LC-MS with a ThermoFinnigan LCQ Deca-XP Plus Ion-Trap Mass Spectrometer (Thermo-FisherScientific Waltham Mass., USA), operated in positive ionization mode.Data dependent scanning was used to generate initial MS to MS² data.Higher order MS^(n) experiments were conducted as required to elucidatestructural information. The mass spectrometer was coupled to a ShimadzuSil HT-C combined autosampler/controller combined with a Shimadzu LC-10Abinary gradient pump system (Shimadzu Scientific Instruments, Columbia,Md., USA). Gradient conditions are described in Table 8. Separation ofCompound I and its metabolites was achieved using a Zorbax XDB C-18 HPLCcolumn (3.0×150 min, 3.5 μm) (Agilent, Santa Clara, Calif. USA) with amobile phase flow rate of 300 μL/min. Mobile phase “A” consisted of 5 mMammonium formate in Millipore water which had been pH adjusted to pH 3.2with formic acid. Mobile phase “B” consisted of 90% acetonitrile/10%methanol.

TABLE 8 HPLC Gradient Elution Scheme Time % Mobile Phase B 0.0 10 1.0 1045.0 40 50.0 90 54.0 90 54.1 10 60.0 10Method A4: Method for Metabolite Characterization of Rat Bile, Plasmaand Urine

Urine, feces, and bile samples from rats administered Compound I werepooled. Approximately 30% of each sample was pooled from each time pointsuch that 90% of the excreted dose from one rat was contained in asingle sample. Samples from each rat were analyzed separately. Fecessamples were extracted using acetonitrile and extraction recoverydetermined by liquid scintillation counting (LSC) of the extract andcombustion and LSC of the unextracted pellet. When available, metabolitestandards were used to confirm structures. Peaks accounting for <5% ofthe administered dose were not reported unless noted otherwise.

Urine, feces, bile and/or plasma samples were analyzed by HPLC and massspectroscopy to determine the molecular weight of any radioactivelylabeled metabolites and obtain structural information of thesemetabolites.

Method A5: Isolation Method

Urine samples from Compound I pharmacokinetic and toxicokinetic studieswere pooled together from individual animals of the same species, i.e.dog or rat. Urine samples were extracted with ethyl acetate at a volumeratio of 1 to 1 three times. The ethyl acetate extraction fractions werepooled and volatiles were removed using a rotavap. The resulting residuewas dissolved in acetonitrile:water (1:1 v/v), and the solution wassubjected to preparative HPLC purification. Isolation of each metabolitewas achieved by using the three HPLC conditions below in sequentialorder.

The concentrated extract was purified by using a mobile phase consistingof 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (SolventB), on a Zobax SB C18 column (19×150 mm, 5 Tm) using a gradient of 5% to60% solvent B over 20 minutes, and a flow rate of 15 mL/min.

-   -   1. The fractions collected from the first HPLC purification was        then further purified by using a mobile phase consisting of 0.1%        TFA in water (Solvent A) and methanol (Solvent B), on a column        Zobax SB C18 column (9.6×150 mm, 5 Tm) using a gradient of 5% to        95% solvent B over 25 minutes, and a flow rate of 4 mL/min.    -   2. Each fraction collected from the second purification with        [M+H]⁺=321 and 323 was subjected to chiral separation using a        mobile phase consisting of 15% ethanol and 85% hexanes or 30%        ethanol and 70% hexanes, on a chiral column (ChiralCel OD-H,        20×250 mm, 5 Tm) with a flow rate of 15 mL/min.        Method A6: Analysis Method

The retention times for 35, 37 and 38 were obtained from the HPLCanalysis using Zorbax SB C18 column (4.6×150 mm, 80 Å, 3.5 Tin) at acolumn temperature of 40° C. using a mobile phase consisting of 0.05%TFA in Water (Solvent A) and 0.05% TFA in Acetonitrile (Solvent B) witha flow rate of 1 mL/min using a gradient elution scheme shown in Table9. An in-line UV detection was performed at 220 nm.

TABLE 9 Gradient Elution Scheme Time (min) % ACN 0 5 15 95 18 95 18.5 524 5Method A7: Alternate Analysis Method

The retention times for 40, 41 and 42 were obtained from the HPLCanalysis using a Waters Atlantis® T-3 HPLC column (4.6×150 mm, 3.5 nm)(Waters Corporation, Milford, Mass., USA) with a mobile phase flow rateof 400 μL/min. Mobile phase “A” consisted of 5 mM ammonium formate inMillipore water which had been pH adjusted to pH 3.2 with formic acid.Mobile phase “B” consisted of 100% methanol. The initial mobile phasecondition was 90% mobile phase “A”/10% mobile phase “B” with a stepgradient to 27% mobile phase “B” in one minute. The initial gradientthen progressed in a linear manner to 52% mobile phase “B” in 57minutes, followed by a second linear gradient to 95% mobile phase “B” in10 minutes. A five minute column washout period at 95% mobile phase “B”ensued and was followed by a return to starting conditions and an 8minute column re-equilibration prior to the next analytical injection.100% of column eluant was routed through a Shimadzu fixed wavelength UVdetector, SPD-10Avp (Shimadzu Scientific Instruments, Columbia, Md.,USA) monitoring λ 254 nm. Upon exiting the UV detector, the eluantstream was split using a PEEK tee to allow approximately 100 μl of theeluant to be introduced via electrospray ionization to the massspectrometer. The electrospray source voltage was set at 4.5 kV, with acapillary temperature of 325° C., sheath and sweep gasses were set at 50and 30 (arbitrary units), respectively. Initial data dependent MS/MSsettings included an isolation width of 2.0 amu and a collision energysetting of 42. All other instrument settings and potentials wereoptimized to achieve maximum signal to noise ratio for Compound I.

Method A8: Enzymatic Hydrolysis of Glucuronide Metabolites of CompoundI.

Isolated glucuronide conjugate metabolites of Compound I were incubatedwith β-glucuronidase with the reaction proceeding to completion in allcases, yielding the associated aglycone, which was analyzed using thesame LC-MS method as described above. The retention times and massspectra of the liberated aglycones were compared with the retentiontimes and mass spectra of the singly hydroxylated metabolite standardsof Compound I to assign the identity of the conjugate.

Example B

Metabolite samples of molecular weight of 323 were presumed to behydroxyl containing metabolites and were dissolved in 200 μL of CD₃OD,obtained from Isotec. Samples of molecular weight 321 were presumed tocontain a ketone moiety and were dissolved in 200 μL of CDCl₃, obtainedfrom Isotec. Samples which were of a molecular weight other than thatdescribed above were dissolved in 200 μL of DMSO-d₆, also obtained fromIsotec. After dissolution, each sample was placed into a 3 mm NMR tube,obtained from Wilmad Glass. Samples were prepared immediately prior toanalysis.

Samples were analyzed using a Varian INOVA NMR spectrometer operating at500 MHz for ¹H and 125 MHz for ¹³C. A 3 mm triple resonance inversedetection gradient probe was used. The sample was kept at 30° C. duringthe time that all data acquisition was taking place. The probe waslocked and shimmed for each sample. Other NMR experiments were performedto obtain the data necessary to determine the structure.

Compound 31 ¹H (500 MHz, DMSO-D₆): δ 8.58 (s, 1H), 8.23 (s, 1H), 4.47(td, 1H), 3.56 (s, 2H), 3.18 (dd, 1H), 3.13 (dd, 1H), 2.35 (m, 1H), 1.79(m, 2H), 1.59-1.39 (m, 4H), 1.29 (m, 2H).

Compound 32 ¹H (500 MHz, DMSO-D₆): δ 9.24 (s, 1H), 8.66 (s, 1H), 4.56(td, 1H), 3.82 (s, 2H), 3.17 (dd, 1H), 3.11 (dd, 1H), 2.35 (m, 1H),1.79-1.12 (m, 4H), 1.61-1.45 (m, 4H).

Compound 35 ¹H (500 MHz, DMSO-D₆): δ 12.25 (s, 1H), 8.82 (s, 1H), 8.73(s, 1H), 8.37 (s, 1H), 7.64 (m, 1H), 7.08 (m, 1H), 4.93 (dd, J=11.6,3.3, 1H), 3.76 (d, J=4.8, 1H), 3.52 (dd, J=17.2, 11.7, 1H), 3.08 (dd,J=17.4, 3.6), 2.04 (m, 1H), 1.76 (m, 1H), 1.63 (m, 1H), 1.54 (m, 2H),0.95 (m, 1H).

Compound 37 ¹H (500 MHz, CDCl₃): δ 8.71 (s, 1H), 8.17 (s, 1H), 8.00 (s,1H), 4.22 (td, J=9.6, 3.7, 1H), 3.71 (s, 2H), 3.09 (dd, J=17.0, 9.1,1H), 2.91 (dd, J=16.8, 3.7, 1H), 2.55 (m, 1H), 1.94 (m, 1H), 1.80-1.45(m, 5H), 1.27 (m, 1H), 1.19 (m, 1H).

Compound 38 ¹H (500 MHz, DMSO-D₆): δ 11.35 (s, 1H), 8.60 (s, 1H), 8.54(s, 1H), 8.09 (s, 1H), 4.55 (td, J=9.8, 3.8, 1H), 4.10 (m, 1H), 3.79 (s,2H), 3.19 (dd, J=16.9, 9.5, 1H), 3.13 (dd, J=17.5, 4.5, 1H), 2.39 (m,1H), 2.01 (m, 1H), 1.58 (m, 1H), 1.48-1.07 (m, 4H).

Example C

Activity data for Metabolites 1-39, along with free fraction andintrinsic clearance data, can be compared with that for the parentcompound, Compound I. JAK activity assays, free fraction assays, andintrinsic clearance assays are described below. Data points can beobtained for individual stereoisomers of Metabolites 1-39. Themetabolites can be potent inhibitors of JAK1, JAK2, and JAK3, likeCompound I.

In Vitro JAK Kinase Assay

Compounds described herein herein can be tested for inhibitory activityof JAK targets according to the following in vitro assay described inPark et al., Analytical Biochemistry 1999, 269, 94-104. The catalyticdomains of human JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3(a.a. 781-1124) with an N-terminal His tag can be expressed usingbaculovirus in insect cells and purified. The catalytic activity ofJAK1, JAK2 or JAK3 can be assayed by measuring the phosphorylation of abiotinylated peptide. The phosphorylated peptide can be detected byhomogenous time resolved fluorescence (HTRF). IC₅₀s of compounds can bemeasured for each kinase in the reactions that contain the enzyme, ATPand 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mMDTT, and 0.1 mg/mL (0.01%) BSA. The ATP concentration in the reactionscan be 90 μM for Jak1, 30 μM for Jak2 and 3 μM for Jak3. Reactions canbe carried out at room temperature for 1 hr and then stopped with 20 μL45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer,Boston, Mass.). Binding to the Europium labeled antibody can be for 40minutes and HTRF signal can be measured on a Fusion plate reader (PerkinElmer, Boston, Mass.). Compounds having an IC₅₀ of 10 μM or less for anyof the above-mentioned JAK targets can be considered active.

Free Fraction Assay

The protein binding of a test compound can be determined by equilibriumdialysis using a Dianorm system from Harvard Apparatus (Holliston,Mass.). The dialysis can be performed at 37° C. for 2 hrs in humanserum. The metabolites can be incubated at 3 μM, and Compound I at 3 and10 μM. The compound concentrations in serum and buffer post-dialysis canbe determined by LC/MS/MS analysis. Free fraction is defined as theratio of the buffer versus serum concentration.

Intrinsic Clearance Assay

Intrinsic clearance can be determined by incubating 1 μM of testcompound in human mixed gender liver microsomes (0.5 mg/mL protein) at37° C. in the presence of 1 mM NADPH. The disappearance of the testcompound can be monitored by LC/MS at 0, 5, 10, 20 and 30 min. The slopeof decline in compound concentration can be used to calculate the humanintrinsic clearance by employing standard methods reported in theliterature.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

What is claimed is:
 1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: n C—H groups areeach independently replaced with C—OH; or, one CH₂ group isindependently replaced with a C═O; and n is 1, 2, 3, or 4; provided thatthe compound is not selected from:3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxocyclopentyl)propanenitrile;and pharmaceutically acceptable salts thereof.
 2. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein: the carbonatom alpha to the cyano group is not replaced with a C—OH or C═O group;and the carbon atom beta to the cyano group is not replaced with a C—OHgroup.
 3. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein n C—H groups are each independently replaced with C—OH.4. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein n is
 1. 5. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein n is
 2. 6. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein n is
 3. 7. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein n is
 4. 8. The compound of claim 1, ora pharmaceutically acceptable salt thereof, wherein one CH₂ group isindependently replaced with a C═O.
 9. A compound of claim 1, selectedfrom:3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1,2-dihydroxycyclopentyl)propanenitrile;and3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;or a pharmaceutically acceptable salt of any of the aforementioned. 10.A compound of claim 1, selected from:3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-3-hydroxypropanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-2-hydroxypropanenitrile;3-cyclopentyl-3-(5-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1,2-dihydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1,3-dihydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-2-hydroxy-3-(1-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-hydroxy-3-(1-hydroxycyclopentyl)propanenitrile;3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;3-(4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;3-(4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;3-(4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;3-(5-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(1-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-2-hydroxy-3-(2-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2,3-dihydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2,4-dihydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2,5-dihydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-hydroxy-3-(2-hydroxycyclopentyl)propanenitrile;3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;3-(4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;3-(4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;3-(4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;3-(5-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-2-hydroxy-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3,4-dihydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-hydroxy-3-(3-hydroxycyclopentyl)propanenitrile;3-(5-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentyl-2,3-dihydroxypropanenitrile;3-cyclopentyl-3-hydroxy-3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-hydroxy-3-(4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-hydroxy-3-(4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-hydroxy-3-(4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-hydroxy-3-(5-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-2-hydroxy-3-(3-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-2-hydroxy-3-(4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-2-hydroxy-3-(4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-2-hydroxy-3-(4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-2-hydroxy-3-(5-hydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(3,5-dihydroxy-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(3-hydroxy-4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(3-hydroxy-4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(3-hydroxy-4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(4-(2,5-dihydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(4-(2,6-dihydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(4-(5,6-dihydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(5-hydroxy-4-(2-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(5-hydroxy-4-(5-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(5-hydroxy-4-(6-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;2-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-2-cyclopentylacetylcyanide; and3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-oxocyclopentyl)propanenitrile;or a pharmaceutically acceptable salt of any of the aforementioned. 11.A compound selected from:3-cyclopentyl-3-(4-(2,6-dioxo-3,5,6,7-tetrahydro-2H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(4-(2-hydroxy-6-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-cyclopentyl-3-(4-(6-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;3-(3-hydroxycyclopentyl)-3-(4-(6-oxo-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;and3-cyclopentyl-3-(4-(5,6-dihydroxy-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile;or a pharmaceutically acceptable salt of any of the aforementioned. 12.The compound of claim 1, or pharmaceutically acceptable salt thereof,which is substantially isolated.
 13. A composition comprising a compoundof claim 1, or a pharmaceutically acceptable salt thereof, and at leastone pharmaceutically acceptable carrier.
 14. The composition of claim13, which is suitable for oral administration.